Aurora kinase inhibitors and methods of making and using thereof

ABSTRACT

Described herein are inhibitors of Aurora kinase and their use in the treatment of cancer. Methods of screening for selective inhibitors of Aurora kinases are also disclosed.

CROSS-REFERENCE TO PRIORITY APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/008,586, filed Nov. 12, 2013, which is a 371 National Phase ofPCT/US2012/031494, filed Mar. 30, 2012, which claims the benefit ofpriority to U.S. Provisional Application No. 61/469,373, filed Mar. 30,2011, and U.S. Provisional Application No. 61/585,091, filed Jan. 10,2012, which are incorporated herein by reference in their entireties.

FIELD

The subject matter disclosed herein relates generally to cancer therapyand to anti-cancer compounds. More specifically, the subject matterdisclosed herein relates to inhibitors of Aurora kinase and their use inthe treatment of cancer. Methods of screening for selective inhibitorsof Aurora kinases are also disclosed.

BACKGROUND

A majority of protein kinases share a common DFG (Asp-Phe-Gly) motif inthe ATP site which transitions between two distinct conformations inresponse to phosphorylation of the contiguous activation loop: theactive DFG-in and the inactive DFG-out states. Kinase inhibitors thatbind only to the DFG-in state often suffer from a lack of targetspecificity, as the ATP site is wide open and able to accommodatediverse chemical scaffolds. By contrast, inhibitors able to induce andstabilize the DFG-out conformation are considered superior, as theyrender the active site architecture incompatible with substrate binding,resulting in enhanced potency and target selectivity. The clinicalsuccess of imatinib (Gleevec) as an inhibitor of Abl kinase (Nagar etal., (2002) Cancer Res 62:4236-4243) is attributed in large part to thisdistinct mode of action (Seeliger et al., (2009) Cancer Res69:2384-2392) and has spurred the design of DFG-out inhibitors for otherkinases, including MAP (Angell et al., (2008) Bioorg Med Chem Lett18:4433-4437), JNK2 (Kuglstatter et al., (2010) Bioorg Med Chem Lett20:5217-5220), Nek2 (Solanki et al., (2011) J Med Chem 54:1626-1639),and Eph receptor tyrosine kinase (Choi et al., (2009) Bioorg Med ChemLett 19:4467-4470). However, all known Aurora kinase inhibitors, such asthe aforementioned chemical probe VX680, are DFG-in inhibitors. Althoughthe DFG motif is highly conserved among protein kinases, the mechanismby which small molecules induce the DFG flip is not well understood.Small molecules able to induce large conformational changes in thetarget enzyme have potential as superior lead compounds in drugdiscovery, as the altered structure of the dead-end complex is lesssuited for efficient interaction with substrate. This concept has led tothe design of some of the most clinically successful kinases inhibitorsto date. Imatinib and sorafenib stabilize the DFG-out conformation byestablishing a bridging network of hydrogen bonds between the amide/ureainhibitor core and both a conserved glutamate side chain within theC-helix and the main chain amide of the DFG aspartate residue (Dietrichet al., (2010) Bioorg Med Chem 18:5738-5748). Molecular dynamicssimulations were used to elucidate and propose a mechanism for theDFG-out conformation in MAPK p38a, in which the phenylalanine of the DFGmotif is forced by the inhibitor from its hydrophobic pocket in theDFG-in (active state) to the solvent-exposed DFG-out (inactive state),triggering an overall rearrangement of the activation loop (Filomia etal., (2010) Bioorg Med Chem 18:6805-6812). However, the knowledge gainedfrom these structures did not translate into an applicable method forthe rational design of DFG-out inhibitors of other kinases.

DFG-out inhibitors of Aurora A utilizing a bisanilinopyrimidine scaffoldare disclosed herein. A series of co-crystal structures established thatelectronegative and electron-withdrawing substituents, directed at theN-terminally flanking residue Ala273, yielded highly potent DFG-outinhibitors able to induce and stabilize a unique “DFG-out/loop-in”conformation. The data suggest an unprecedented mechanism of action, bywhich induced-dipole forces disrupt the charge distribution along theDFG peptide, causing the DFG to unwind. As the ADFG sequence is highlyconserved among kinases, the strategy employed here to inhibit Aurora Amay be applicable to other kinases as well.

SUMMARY

In accordance with the purposes of the disclosed materials and methods,as embodied and broadly described herein, the disclosed subject matter,in one aspect, relates to compounds, compositions and methods of makingand using compounds and compositions. In specific aspects, the disclosedsubject matter relates to cancer therapy and to anti-cancer compounds.More specifically, the subject matter disclosed herein relates toinhibitors of Aurora kinase and their use in the treatment of cancer.Further, disclosed herein are DFG-out inhibitors of Aurora kinase.Methods of screening for new Aurora kinase inhibitors are alsodisclosed.

Additional advantages will be set forth in part in the description thatfollows, and in part will be obvious from the description, or may belearned by practice of the aspects described below. The advantagesdescribed below will be realized and attained by means of the elementsand combinations particularly pointed out in the appended claims. It isto be understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the United States Patent andTrademark Office upon request and payment of the necessary fee.

FIG. 1A shows compound 1, a potent and highly selective inhibitor forAurora A over Aurora B, and its interactions with Aurora A. Syntheticalternatives to various functional groups on the molecule are alsodescribed. Specifically, FIG. 1A shows the structure of compound 1. FIG.1B shows the specific interactions of compound 1 with various residuesof Aurora A. FIG. 1C describes various synthetic modifications ofcompound 1.

FIG. 2A is a drawing that shows compound 1's interactions with Aurora A.FIG. 2A shows the specific interactions of compound 1 with variousresidues of Aurora A (also shown in FIG. 1(b)). FIG. 2B shows the X-raystructure of compound 1 bound to Aurora A active site (compound 1). FIG.2C shows compound 1 co-crystallized with Aurora A. The hinge region,DFG-in activation loop open conformation (compound 1). FIG. 2D is astructural overlay of ADP and compound 1. FIG. 2E shows ADP bound toAurora A active site.

FIG. 3 is a general synthetic route to diphenylpyrimidine-2,4-diaminecompounds. Reagents and conditions; method a: compound 1 is HLM008598shown in FIG. 1, 0.1 M HCl in water (1-3 mL/mmol), microwave, 100° C.,30 min-24 h., 30-90% (when R³═Cl, DIPEA, n-BuOH, seal tube, 150° C.,reflux 4 days, 43%); method b: 0.1 M HCl in water (1.5 or 3 mL/mmol),microwave, 160° C., 15 min., 21-97%; method c: EtOH/1 M HCl water (1/1,4 or 12 mL/mmol), microwave, 160° C., 15 min-1 h; method d: 4 M HCl inDioxane (0.5 mL/mmol), 2-butanol (3 mL/mmol), seal tube, 120° C.,overnight (24 h); method e: THF/1 M HCl water (1/2, 6 mL/mmol),microwave, 160° C., 15 min.); method f. THF/2 M NaOH water solution(1/2, 4 mL/mmol), seal tube, 100° C., 0.5, 69%.

FIG. 4A is a synthetic route to a bisanilinopyrimidine carboxylic acidlibrary, which is shown in FIG. 4B and in FIG. 4C. Reagents andconditions; method a: HCl (0.1 M, aq., 1-3 mL/mmol), microwave, 100° C.,30 min. method b: HCl (0.1 M, aq., 3 mL/mmol), r.t., 3-5 days. method c:HCl (0.1 M, aq., 1.5 mL/mmol), sealed tube, 100° C., 24 h. method d: HCl(0.1 M, aq., 3-6 mL/mmol), microwave, 160° C., 15 min. method e: EtOH:HCl (1 M, aq., 1:1, 4 mL/mmol), microwave, 160° C., 15 min.-1 h. methodf (i) HCl (4 M in dioxane, 0.5 mL/mmol), 2-butanol (3 mL/mmol), sealedtube, 120° C., overnight (24 h), 72% or (ii) EtOH, sealed tube, 120° C.,overnight—4 days. method g: THF: HCl (1 M, aq.) (1:2, 6 mL/mmol),microwave, 160° C., 15 min. method h: THF:NaOH (2 M, aq.) (1:2, 4-7mL/mmol), sealed tube, 85-100° C., 0.5-16 h.

FIG. 5A is a synthetic route to bisanilinopyrimidine library 6 withhalogens (F, Cl, Br, and I), CN, non-polar groups (H, Ph) and polarhydrophobic groups (OCF₃, CF₃ and OMe) on the A-ring, which library isshown in FIG. 5B and in FIG. 5C. Reagents and conditions: method i:n-BuOH, DIPEA, 125° C. method j: n-BuOH, Na₂CO₃, 100° C. method k DMF,NaH, r.t., overnight (14-16 h). method l: EtOH (1 drop of 1M HCl),microwave, 160° C., 15 min., 56-75%, method m: EtOH or MeOH, 150° C., 20min., microwave. method n: Cat. HCl, THF, reflux 14 h. method o: THF,NaOH (1.8 M aq., 5 equivalents), THF, reflux, 14 h., method b: HCl (0.1M, aq., 3 mL/mmol), r.t., 3-5 days.

FIG. 6 is a synthetic route to building blocks with water solubilizinggroups. Reagents and conditions: method p: 10% Pd/C, EtOH, H₂, r.t.,overnight; method q: H-Cube, 10% Pd/C, MeOH, 30 bar, 4 Loops, rt.

FIG. 7A is a synthetic route to bisanilinopyrimidines withwater-solubilizing groups in the B-ring. Various derivatives are alsoshown in FIG. 7B. Reagents and conditions; method r: Conc. HCl,iso-PrOH, 170° C., microwave, 20 min., method s: EtOH, 150° C.,microwave, 20 min., method t: EtOH, cat. HCl, 180° C. or 160° C. or 140°C. microwave, 15 min., method u: X-Phos (10 mmol %),bis(dibenzylideneacetone) palladium(0) (10 mmol %), K₂CO₃, tert-BuOH,reflux, 18 h., method v: MeOH, 100° C., seal tube, 6 h.

FIG. 8 is a synthetic route to bisanilinopyrimidine tetrazolederivatives. Reagents and conditions: method a: 12a: Ethanol, microwave,150° C., 40 min., 37%; 12b: Ethanol, microwave, 170° C., 40 min., 33%;12c: Ethanol, microwave, 160° C., 40 min., 36%; 12d: Ethanol, microwave,150° C., 40 min., 25%; method b: 10a and 10b: NaN₃, Et₃N.HCl, toluene,100° C., 15 h, 92% and 95%; method c: 11a and 11b: H₂, Pd/C, methanol,r.t., 20 h, 93% and 98%.

FIG. 9 illustrates compound 3l with DGF activation loop closed bound toAurora A.

FIG. 10A, FIG. 10B and FIG. 10C illustrate a comparison of ATP,compounds 1 and 3l bound to Aurora A crystal structure. FIG. 10Aillustrates ATP bound to Aurora A. FIG. 10B illustrates compound 1 boundto Aurora A. FIG. 10C illustrates compound 3l bound to Aurora A.

FIG. 11A and FIG. 11B illustrate a comparison of surface structures forcompounds 1 (FIG. 11A) and 3l (FIG. 11B) with Aurora A.

FIG. 12A through FIG. 12D illustrate the X-ray structures of compounds3l (FIG. 12A), 3g (FIG. 12B) and compound 14 (FIG. 12C) bound to AuroraA active site. FIG. 12D shows compound 1 bound to Aurora A; R¹ hydrogenis shown near Leu194, Leu210 and Ala160, which are shown as spheres toshow the narrow space R¹ is occupying in the binding region.

FIG. 13A through FIG. 13C illustrates the inhibition of phosphorylationof serine 10 on Histone H3 (substrate for Aurora a) in MDA-MB-468 cellsby bisanilinopyrimidines with water solubilizing moieties. Inhibition at2 concentrations (1 and 10 μM) for each compound is shown.

FIG. 14A through FIG. 14I show the binding modes of variousbisanilinopyrimidine inhibitors (compounds 3h, 1, 6q, 6h, 6d, 6a, 3l,6i, and 6n) with Aurora A. Crystal structures were determined for AuroraA liganded with different ortho-substituted bisanilinopyrimidineinhibitors (Table 6). The hinge region (residues 211-213) is indicatedlight grey, the DFG (residues 274-276) in darker grey, the activationloop (residues 277-293) in darkest grey. The dotted lines indicate theclosest distances to the DFG. The 2F_(o)-F_(c) electron density,contoured at 1σ, is shown as blue mesh around the inhibitor; theF_(o)-F_(c) electron density maps from refinements omitting theinhibitor are shown in the supporting material (FIG. 23). The insets inthe top right corners are surface representations of the overallstructures. Compounds 3h, 1, 6q, 6h, and 6d are DFG-in inhibitors;compounds 6a, 3l, 6i, and 6n are DFG-out inhibitors.

FIG. 15A through FIG. 15E show structural changes in Aurora A that areinduced by DFG-out inhibitors. FIG. 15A is a surface representation ofAurora A in the DFG-in state (left, liganded with compound 3h) andDFG-out state (right, liganded with compound 3l); the activation loop ishighlighted and the inhibitors are shown. FIG. 15B is a superpositionthat reveals global conformational changes upon binding of compound 7particularly of the activation loop and the C-helix, which harbors thecatalytic residue Glu181. In the DFG-in state, the loop is oriented awayfrom the ATP site and the inhibitor is exposed to solvent. In theDFG-out state, the loop flips by about 180° and the N-terminal flank ispositioned above the active site, shielding the inhibitor from solvent.FIG. 15C shows the conformation of the ADFGW segment in the DFG-in stateliganded with compound 3h and the DFG-out state liganded with compound7. The residue closest to the inhibitor is Ala273 (3.4 Å). The DFG-flipcauses drastic conformational changes of the backbone, beginning withresidue Asp274, forcing Trp277 and the entire activation loop to changedirection. The binding interactions of Trp277 in the DFG-in and DFG-outstates are shown in FIG. 26. The C-helix of FIG. 15B gives way toaccommodate the new conformation of Phe275. FIG. 15D shows that, in theDFG-out conformation, the side chain of Asp274 interacts with residuesArg255 and Asp256, and the conformation of the activation loop isstabilized through hydrogen bonding interactions between the main chainatoms of His280 and Lys141. The loop is shaded according to temperaturefactors from light grey (low B-factor) to dark grey (high B-factor).Potential hydrogen bonding interactions are indicated as black dottedlines. FIG. 15E is a comparison of the molecular mode of action of VX680(PDB 3E5A), compound 3h, and compound 3l (stereo presentation).

FIG. 16A through FIG. 16C show substitutions in other regions of thebisanilinopyrimidine scaffold do not affect the DFG-out mode of action(stereo presentations). FIG. 16A shows compounds 3o and 13a areanalogues of the DFG-out inhibitor compound 3l (substitutions arehighlighted). Both inhibitors induced the DFG flip and displayed thesame general interaction pattern as compound 3l. FIG. 16B shows thatintroduction of a fluorine to the pyrimidine ring (compound 3o) fostersvan-der-Waals interactions with hydrophobic residues around thegatekeeper residue Leu210, resulting in increased inhibitory activity.FIG. 16C shows that substitution of tetrazole for carboxyl inpara-position of the B-ring (compound 3a) preserves the electrostaticinteraction with Arg137, and the inhibitory potency remains unchanged.Shown in blue mesh is the 2F_(o)-F_(c) electron density of theinhibitors, contoured at 1σ. Potential hydrogen bonding and hydrophobicinteractions are indicated as dotted lines.

FIG. 17A through FIG. 17C show the dipole-induced mechanism of actionfor Aurora A DFG-out inhibitors. FIG. 17A shows a model of the collisioncomplex of the DFG-in state of Aurora A with the DFG-out inhibitorcompound 3l, based on superimposition of the co-crystal structures ofcompounds 3l and 3h. Displayed are the closest distances (Å) between thechlorine substituent and the enzyme. The ˜0.8 Å reduced distance in thedead-end complex indicates attraction of Ala273, a feature observed forthe DFG-out inhibitors compounds 6a, 3l, 6i, and 6n and, to a lesserdegree, for the DFG-in inhibitors compounds 6h and 6d (FIGS. 21 and 22).FIG. 17B is a schematic showing the electric dipoles along the C—R bonds(R═F, Cl, Br, C≡N) of the inhibitor can induce a dipole along theC_(α)-C_(β) bond of Ala273. The dipole-dipole interaction is stabilizedby altering the charge distribution along the DFG backbone, allowing orforcing the compact DFG-in state to unwind. FIG. 17C shows a geometricarrangement of compounds 6h, 6d, 6a, 3l, 6i, and 6n and Ala273 in theexperimentally determined dead-end complexes. Substituents able toinduce the DFG flip (compounds 6a, 3l, 6i, and 6n) align linearly withthe C_(α)-C_(β) bond of Ala273, whereas the C—F bonds of the DFG-incompounds 6h and 6d are positioned orthogonal.

FIG. 18A through FIG. 18E show the implications for the design ofDFG-out inhibitors of other kinases. FIG. 18A shows the ADFG-in statesof Aurora A, ABL1, Rock1, and LCK are highly similar, indicating thatexogenous dipoles directed at the alanine residue can induce similarstructural changes in these kinases. FIG. 18B shows that CDK2, MAPK3,and MER adopt a different conformation in the C-terminal flank andtherefore can respond to exogenous dipoles differently. The r.m.s.d.values for the ADFG-in state of various kinases with respect to Aurora Aare shown in Table 2. FIG. 18C shows the conformation of compound 3lbound to the active site of Aurora A is incompatible for efficientbinding with CDK2. The model was generated by superimposition of thecomplexes of Aurora-with compound 3l and CDK2 with compound 3l. FIG. 18Dshows the co-crystal structure of CDK2 in complex with compound 3l andreveals that the bisanilinopyrimidine scaffold adopts an (s)-transconformation (defined as the position of the groups colored red acrossthe C—N bond), the A-ring pointing away from the DFG. FIG. 18E shows the(s)-cis and (s)-trans conformation of compound 3l found in Aurora A andCDK2, respectively. The 2F_(o)-F_(c) electron density around theinhibitor in Panel (d) is contoured at 1σ. Potential hydrogen bonding,van-der-Waals interactions, and steric clashes are indicated as dottedlines. The structure of CDK2 with compound 3lh is shown in the FIG. 25.

FIG. 19 is a graph showing IC₅₀ determination of bisanilinopyrimidineinhibitors with Aurora A.

FIG. 20A through FIG. 20E show binding studies of bisanilinopyrimidineinhibitors with Aurora A by ITC.

FIG. 21A through FIG. 21D show comparisons of the DFG-in dead-endcomplexes (stereo presentations). The structures of compounds 1, 6q, 6h,and 6d were aligned with the structure of 3h. Shown are the inhibitors,the ADFG segment, Lys162 and distances in A. (FIG. 21A) Aurora A—1,(FIG. 21B) Aurora A—6q, (FIG. 21C) Aurora A—6h, (FIG. 21D) Aurora A—6d.

FIG. 22A through FIG. 22 D show comparisons of the DFG-out dead-endcomplexes (stereo presentations). The structures of 1, 6q, and 6h werealigned with the structure of 3h. Shown are the inhibitors, the ADFGsegment, Lys162 and distances in Å. (FIG. 22A) Aurora A—6a, (FIG. 22B)Aurora A—3l, (FIG. 22C) Aurora A—6i, (FIG. 22D) Aurora A—6n.

FIG. 23A through FIG. 23I show binding modes of bisanilinopyrimidineinhibitors with Aurora A. Crystal structures were determined for AuroraA liganded with different ortho-substituted bisanilinopyrimidineinhibitors. The hinge region (residues 211-213) is indicated in lightgrey, the DFG (residues 274-276) in darker grey, the activation loop(residues 277-293) in darkest grey. The dotted lines indicate the closedistance of electronegative groups to the methyl group of Ala273. TheF_(o)-F_(c) electron density resulting from refinement omitting theinhibitor is shown as mesh, contoured at 2.5 σ. The insets are surfacerepresentations of the overall structures. Compounds 3h, 1, 6q, 6h, and6d are DFG-in inhibitors; compounds 6a, 3l, 6i, and 6n are DFG-outinhibitors.

FIG. 24A and FIG. 24B show the crystal structures of 3o and 13a bound toAurora A (Stereo presentations of the binding interactions between theDFG-out inhibitors 3o (FIG. 24A) and 13a (FIG. 24B) to Aurora A. TheF_(o)-F_(c) electron density map of the omitted inhibitor is contouredat 2.5 σ and shown in mesh. Potential hydrogen bonding and van der Waalsinteractions are indicated as dotted lines.

FIG. 25A and FIG. 25B show the co-crystal structures of Aurora Ainhibitors with CDK2. FIG. 25A is the structure of 3h bound to CDK2.FIG. 25B is the structure of compound 3l bound to CDK2. Shown as mesh isthe F_(o)-F_(c) electron density map from refinements omitting theinhibitors, contoured at 2.5 σ.

FIG. 26A shows that in the DFG-in state with compound 3h, the main chaincarbonyl oxygen of Trp277 forms hydrogen bonding interactions with theguanidinium group of Arg255 (black dotted lines). The indole moiety issurrounded by mostly polar residues with weak potential for VDWinteractions (dotted lines). FIG. 26B shows that in the DFG-out statewith compound 3l, the indole moiety is positioned in a strictlyhydrophobic pocket with high VDW interaction potential; polarinteractions between Trp277 and neighboring residues no longer exist.

DETAILED DESCRIPTION

The materials, compounds, compositions, and methods described herein maybe understood more readily by reference to the following detaileddescription of specific aspects of the disclosed subject matter, theFigures, and the Examples included therein.

Before the present materials, compounds, compositions, and methods aredisclosed and described, it is to be understood that the aspectsdescribed below are not limited to specific synthetic methods orspecific reagents, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.

Also, throughout this specification, various publications arereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which the disclosed matterpertains. The references disclosed are also individually andspecifically incorporated by reference herein for the material containedin them that is discussed in the sentence in which the reference isrelied upon.

General Definitions

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

Throughout the specification and claims the word “comprise” and otherforms of the word, such as “comprising” and “comprises,” means includingbut not limited to, and is not intended to exclude, for example, otheradditives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a composition”includes mixtures of two or more such compositions, reference to “aninhibitor” includes mixtures of two or more such inhibitors, referenceto “the kinase” includes mixtures of two or more such kinase, and thelike.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Furthermore, when numerical ranges ofvarying scope are set forth herein, it is contemplated that anycombination of these values inclusive of the recited values may be used.Further, ranges can be expressed herein as from “about” one particularvalue, and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. Unless stated otherwise, the term “about” means within 5%(e.g., within 2% or 1%) of the particular value modified by the term“about.”

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant lowering of an event or characteristic (e.g.,tumor growth, metastasis). It is understood that this is typically inrelation to some standard or expected value, in other words it isrelative, but that it is not always necessary for the standard orrelative value to be referred to. For example, “reduces tumor growth”means decreasing the amount of tumor cells relative to a standard or acontrol.

By “prevent” or other forms of the word, such as “preventing” or“prevention,” is meant to stop a particular event or characteristic, tostabilize or delay the development or progression of a particular eventor characteristic, or to minimize the chances that a particular event orcharacteristic will occur. Prevent does not require comparison to acontrol as it is typically more absolute than, for example, reduce. Asused herein, something could be reduced but not prevented, but somethingthat is reduced could also be prevented. Likewise, something could beprevented but not reduced, but something that is prevented could also bereduced. It is understood that where reduce or prevent are used, unlessspecifically indicated otherwise, the use of the other word is alsoexpressly disclosed.

As used herein, “treatment” refers to obtaining beneficial or desiredclinical results. Beneficial or desired clinical results include, butare not limited to, any one or more of: alleviation of one or moresymptoms (such as tumor growth or metastasis), diminishment of extent ofcancer, stabilized (i.e., not worsening) state of cancer, preventing ordelaying spread (e.g., metastasis) of the cancer, preventing or delayingoccurrence or recurrence of cancer, delay or slowing of cancerprogression, amelioration of the cancer state, and remission (whetherpartial or total).

The term “patient” preferably refers to a human in need of treatmentwith an anti-cancer agent or treatment for any purpose, and morepreferably a human in need of such a treatment to treat cancer, or aprecancerous condition or lesion. However, the term “patient” can alsorefer to non-human animals, preferably mammals such as dogs, cats,horses, cows, pigs, sheep and non-human primates, among others, that arein need of treatment with an anti-cancer agent or treatment.

It is understood that throughout this specification the identifiers“first” and “second” are used solely to aid in distinguishing thevarious components and steps of the disclosed subject matter. Theidentifiers “first” and “second” are not intended to imply anyparticular order, amount, preference, or importance to the components orsteps modified by these terms.

Chemical Definitions

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a mixture containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the mixture.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc.

The term “aliphatic” as used herein refers to a non-aromatic hydrocarbongroup and includes branched and unbranched, alkyl, alkenyl, or alkynylgroups.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl,tetracosyl, and the like. The alkyl group can also be substituted orunsubstituted. The alkyl group can be substituted with one or moregroups including, but not limited to, alkyl, halogenated alkyl, alkoxy,alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid,ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo,sulfonyl, sulfone, sulfoxide, or thiol, as described below.

The symbols A^(n) is used herein as merely a generic substitutent in thedefinitions below.

The term “alkoxy” as used herein is an alkyl group bound through asingle, terminal ether linkage; that is, an “alkoxy” group can bedefined as —OA¹ where A¹ is alkyl as defined above.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (A¹A²)C═C(A³A⁴)are intended to include both the E and Z isomers. This may be presumedin structural formulae herein wherein an asymmetric alkene is present,or it may be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide,or thiol, as described below.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be substituted with oneor more groups including, but not limited to, alkyl, halogenated alkyl,alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylicacid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo,sulfonyl, sulfone, sulfoxide, or thiol, as described below.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “heteroaryl” isdefined as a group that contains an aromatic group that has at least oneheteroatom incorporated within the ring of the aromatic group. Examplesof heteroatoms include, but are not limited to, nitrogen, oxygen,sulfur, and phosphorus. The term “non-heteroaryl,” which is included inthe term “aryl,” defines a group that contains an aromatic group thatdoes not contain a heteroatom. The aryl and heteroaryl group can besubstituted or unsubstituted. The aryl and heteroaryl group can besubstituted with one or more groups including, but not limited to,alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol asdescribed herein. The term “biaryl” is a specific type of aryl group andis included in the definition of aryl. Biaryl refers to two aryl groupsthat are bound together via a fused ring structure, as in naphthalene,or are attached via one or more carbon-carbon bonds, as in biphenyl.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group asdefined above where at least one of the carbon atoms of the ring issubstituted with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkylgroup can be substituted or unsubstituted. The cycloalkyl group andheterocycloalkyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide,or thiol as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and containing at least onedouble bound, i.e., C═C. Examples of cycloalkenyl groups include, butare not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term“heterocycloalkenyl” is a type of cycloalkenyl group as defined abovewhere at least one of the carbon atoms of the ring is substituted with aheteroatom such as, but not limited to, nitrogen, oxygen, sulfur, orphosphorus. The cycloalkenyl group and heterocycloalkenyl group can besubstituted or unsubstituted. The cycloalkenyl group andheterocycloalkenyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide,or thiol as described herein.

The term “cyclic group” is used herein to refer to either aryl groups,non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl groups), or both. Cyclic groups have one or more ringsystems that can be substituted or unsubstituted. A cyclic group cancontain one or more aryl groups, one or more non-aryl groups, or one ormore aryl groups and one or more non-aryl groups.

The term “aldehyde” as used herein is represented by the formula —C(O)H.Throughout this specification “C(O)” is a short hand notation for C═O.

The terms “amine” or “amino” as used herein are represented by theformula NA¹A²A³, where A¹, A², and A³ can be, independently, hydrogen,an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl,cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groupdescribed above.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH. A “carboxylate” as used herein is represented by the formula—C(O)O—.

The term “ester” as used herein is represented by the formula —OC(O)A¹or —C(O)OA¹, where A¹ can be an alkyl, halogenated alkyl, alkenyl,alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl,or heterocycloalkenyl group described above.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “ketone” as used herein is represented by the formula A¹C(O)A²,where A¹ and A² can be, independently, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “halide” as used herein refers to the halogens fluorine,chlorine, bromine, and iodine.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “cyano” as used herein is represented by the formula —CN

The term “azido” as used herein is represted by the formula —N₃.

The term “sulfonyl” is used herein to refer to the sulfo-oxo grouprepresented by the formula —S(O)₂A¹, where A¹ can be hydrogen, an alkyl,halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group describedabove.

The term “sulfonylamino” or “sulfonamide” as used herein is representedby the formula —S(O)₂NH₂.

The term “thiol” as used herein is represented by the formula —SH.

It is to be understood that the compounds provided herein may containchiral centers. Such chiral centers may be of either the (R—) or (S—)configuration. The compounds provided herein may either beenantiomerically pure, or be diastereomeric or enantiomeric mixtures. Itis to be understood that the chiral centers of the compounds providedherein may undergo epimerization in vivo. As such, one of skill in theart will recognize that administration of a compound in its (R—) form isequivalent, for compounds that undergo epimerization in vivo, toadministration of the compound in its (S—) form.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), nuclearmagnetic resonance (NMR), gel electrophoresis, high performance liquidchromatography (HPLC) and mass spectrometry (MS), gas-chromatographymass spectrometry (GC-MS), and similar, used by those of skill in theart to assess such purity, or sufficiently pure such that furtherpurification would not detectably alter the physical and chemicalproperties, such as enzymatic and biological activities, of thesubstance. Both traditional and modern methods for purification of thecompounds to produce substantially chemically pure compounds are knownto those of skill in the art. A substantially chemically pure compoundmay, however, be a mixture of stereoisomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer, diastereomer, and meso compound,and a mixture of isomers, such as a racemic or scalemic mixture.

A “pharmaceutically acceptable” component is one that is suitable foruse with humans and/or animals without undue adverse side effects (suchas toxicity, irritation, and allergic response) commensurate with areasonable benefit/risk ratio.

“Pharmaceutically acceptable salt” refers to a salt that ispharmaceutically acceptable and has the desired pharmacologicalproperties. Such salts include those that may be formed where acidicprotons present in the compounds are capable of reacting with inorganicor organic bases. Suitable inorganic salts include those formed with thealkali metals, e.g., sodium, potassium, magnesium, calcium, andaluminum. Suitable organic salts include those formed with organic basessuch as the amine bases, e.g., ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, and the like. Suchsalts also include acid addition salts formed with inorganic acids(e.g., hydrochloric and hydrobromic acids) and organic acids (e.g.,acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). When two acidic groups are present, a pharmaceutically acceptablesalt may be a mono-acid-mono-salt or a di-salt; similarly, where thereare more than two acidic groups present, some or all of such groups canbe converted into salts.

“Pharmaceutically acceptable excipient” refers to an excipient that isconventionally useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic, and desirable, and includes excipients thatare acceptable for veterinary use as well as for human pharmaceuticaluse. Such excipients can be solid, liquid, semisolid, or, in the case ofan aerosol composition, gaseous.

A “pharmaceutically acceptable carrier” is a carrier, such as a solvent,suspending agent or vehicle, for delivering the disclosed compounds tothe patient. The carrier can be liquid or solid and is selected with theplanned manner of administration in mind. Liposomes are also apharmaceutical carrier. As used herein, “carrier” includes any and allsolvents, dispersion media, vehicles, coatings, diluents, antibacterialand antifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician. In reference to cancers or other unwanted cellproliferation, an effective amount comprises an amount sufficient tocause a tumor to shrink and/or to decrease the growth rate of the tumor(such as to suppress tumor growth) or to prevent or delay other unwantedcell proliferation. In some embodiments, an effective amount is anamount sufficient to delay development. In some embodiments, aneffective amount is an amount sufficient to prevent or delay occurrenceand/or recurrence. An effective amount can be administered in one ormore doses. In the case of cancer, the effective amount of the drug orcomposition may: (i) reduce the number of cancer cells; (ii) reducetumor size; (iii) inhibit, retard, slow to some extent and preferablystop cancer cell infiltration into peripheral organs; (iv) inhibit(i.e., slow to some extent and preferably stop) tumor metastasis; (v)inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrenceof tumor; and/or (vii) relieve to some extent one or more of thesymptoms associated with the cancer.

Effective amounts of a compound or composition described herein fortreating a mammalian subject can include about 0.1 to about 1000 mg/Kgof body weight of the subject/day, such as from about 1 to about 100mg/Kg/day, especially from about 10 to about 100 mg/Kg/day. The dosescan be acute or chronic. A broad range of disclosed composition dosagesare believed to be both safe and effective.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, articles, and methods,examples of which are illustrated in the accompanying Examples andFigures.

Compounds

Disclosed are compounds that are Aurora kinase inhibitors, e.g., AuroraA, B, and/or C kinase inhibitors. These disclosed compounds can be usedin various compositions as anti-cancer therapeutics.

In certain embodiments, the disclosed compounds have the chemicalstructure shown in Formula I.

In Formula I, R¹ is selected from the group consisting of H, Cl, F, Br,I, C₁-C₆ alkyl, CN, NO₂, and NH₂.

Also in Formula I, R² is H, F, or Cl.

Additionally in Formula I, each R³ is selected, independently, from thegroup consisting of Cl, Br, F, COOH, CF₃, CN, phenyl, OCH₃, COR⁵, CONH₂,CONR⁵, and COONH₂.

Further in Formula I, each R⁴ is selected, independently, from the groupconsisting of H, COOH, CONH₂, CONR⁵, SO₂NH₂, CONSO₂R⁵, tetrazole,4-morpholine, or COR⁵. R⁵ is C₁-C₆ alkyl, cycloalkyl, heteroaryl, orheteroalkyl.

Still further in Formula I, n is 1-5 (e.g., 1, 2, 3, 4, or 5) and m is1-5 (e.g., 1, 2, 3, 4, or 5). Thus, in the disclosed compounds there canbe from 1 to 5 different substituents R³ and from 1 to 5 differentsubstituents R⁴. Pharmaceutically acceptable salts of these compoundsare also disclosed.

In certain preferred aspects, the compound has formula I, wherein R³ is2-Cl, and n is 1. In other examples, the compound has formula I, whereinR⁴ is 4-COOH, and m is 1. In still other examples, the compound hasformula I, wherein R³ is 2-Cl, n is 1, m is 1, and R⁴ is COOH, COR⁵,CONH₂, CONR⁵, or CONSO₂R⁵, wherein R⁵ is C₁-C₆ alkyl, cycloalkyl,heteroaryl, or heteroalkyl.

Still further, the disclosed compounds can have the following formulaII:

In Formula II, R¹ is selected from the group consisting of H, Cl, F, Br,I, CH₃ and NH₂; R² is H, F, or Cl.

Also in Formula II, R³ is selected from the group consisting of 2-Cl,2-Br, 2-F, 2-COOH, 2-CF₃, 2-CN, 2-phenyl, 2-OCH₃, 2-COONH₂, 4-COOH,4-OCH₃.

Additionally in Formula II, R⁴ is selected from the group consisting ofH, COOH, 2-CONH₂, 4-CONH₂, SO₂NH₂, tetrazole, and 4-morpholine.

Still further, the disclosed compounds can have the following formulaIII:

In Formula III, R¹, R², R⁴, and m are as defined herein.

As disclosed herein, a series of compounds have been designed andprepared that, according to the data, have an unprecedented mechanism ofaction to induce the DFG flip in Aurora A, and other kinases as well, byinduced-dipole forces. The vast majority of kinase inhibitors areso-called Type I inhibitors such as compounds 3h, 1, 6q, 6h, and 6d,which compete with ATP for binding to the open DFG-in state. Theunderlying mechanism by which small molecules induce the DFG flip is notunderstood, and experimental data are limited to a few well-studiedcases such as Abl, p38-MAP and MEKI (Schindler et al., (2000) Science289:1938-1942; Pargellis et al., (2002) Nat Struct Biol 9:268-272). Forthese kinases, potent and selective Type II (partially allosteric) andType III (fully allosteric) inhibitors have been designed, which act byoccupying a pocket adjacent to the ATP site (Zhang et al., (2010) Nature463:501-506; Comess et al., (2011) ACS Chem Biol 6:234-244; Dong et al.,(2011) Bioorg Med Chem Lett 21:1315-1319). Although the here designedcompounds 6a, 3l, 6i, 6n, 3o, and 13a are purely Type I, they induce andstabilize the DFG-out state with drastic consequences for the overallstructure of the enzyme. These findings offer new opportunities in therational design of DFG-out inhibitors of various kinases, by equippingsuitable DFG-in inhibitor scaffolds with electric dipoles directed atthe N-terminal flank.

FIG. 3 describes the general synthetic route used for preparation ofdianilinipyrimidine (1) and diphenylpyrimidine-2,4-diamine-focusedlibraries (3) and (4) from readily available building blocks. The2,4-dichloropyrimidine was initially reacted with the requisitecommercially available anilines with the method predominantly usingaqueous hydrochloric acid as the solvent, with microwave-assistedheating to obtain the required analogs (2). The library (2) was furtherfunctionalized to provide the libraries (3) and (4) bearing the B ringin good yields.

The common reported binding mode for kinases is ‘DFG in’, which is theATP binding mode. See FIG. 14A through FIG. 16C. The compound 3h boundto Aurora A was observed in ‘DFG in’ mode, the same as ATP.Interestingly, the co-crystal structure of compound 3m with Aurora Ashowed the ‘DFG out’ conformation, which is rarely observed with AuroraA kinase. The ortho-Cl in A ring in compound 3m induces a largeconformational change of the activation loop, where the ATP binding siteis completely concealed by the closed activation loop.

The screening of compound 1 was identified as a potent and selectiveinhibitor for Aurora A (in vitro IC₅₀=0.073±0.002 μM) over Aurora B (invitro IC₅₀=5.4±1.8 μM). Re-synthesis of(1) confirmed the structure andactivity. The synthetic modifications around (1) are shown in the FIG.1C.

Ortho-substitution on ring A impacts activity. Moving ortho-COOH topara-COOH reduced the activity (compound 3g, entry 8 of Table 1,IC₅₀=0.37 μM). Removing the substitution from ring A did not improve theactivity (compound 3l, entry 7 of Table 1, IC₅₀=0.091 μM). Replacementof ortho-COOH in ring A with Cl improved the inhibitory activity by 2fold (compound 3m, entry 15 of Table 1, IC₅₀=0.037 μM).

Para-substitution on ring B impacts activity. Moving para-COOH to orthoposition decreased the activity (compound 3a, entry 2 of Table 1,IC₅₀=0.65 μM); replacement of para-COOH with ortho-NH₂ completely lostactivity. Replacement of ortho-COOH with morpholine did not lead theimprovement of activity (compound 3c, entry 4 of Table 1, IC₅₀=0.26 μM).

The removal of carboxylic acid from both ring A and ring B resulted inreduced inhibitory activity (compound 3d, entry 5 of Table 1, IC₅₀=1.9μM). The methyl esters on both ring A and ring B showed no inhibitoryactivity. The substitution of the carboxylic acids on both ring A andring B with primary amides retained the activity (compound 3e, entry 6of Table 1, IC₅₀=0.086 μM).

The introduction of fluorine as R¹ on the pyrimidine ring showed 2-foldimprovement of activity (compound 3h, entry 9 of Table 1, IC₅₀=0.039μM). However, CH₃ and NH₂ groups as R¹ did not lead to an improvement inactivity (compounds 3i and 4a, entries 10 and 11 of Table 1). This mayindicate that larger hydrophobic groups or hydrophilic groups are nottolerated in the binding site.

The whole cell study of the pyrimdine library was carried out inMDA-MB-468 cell line. The original hit 1 and the most potent compounds3m did not show inhibition in the cell assay, most probably due to poorsolubility and cell permeability. The presence of carboxylic acid inboth A and B rings showed poor cell activity. The compounds 3l, 3n, 3oand 3p (entries 14, 16, 17, 18 of Table 1) showed IC₅₀ less than 20 μMin whole cells.

In summary, Aurora A inhibitors with good activity have been identified.The X-ray crystallography studies assisted to understand the bindingmodes of the compounds under investigation. The conformational changeobserved with compound 3m (the activation loop closed) for Aurora A isan important observation in this study that has not been revealed withthis class of compounds toward Aurora A.

Method

Further provided herein are methods of treating or preventing cancer ina subject, comprising administering to the subject an effective amountof a compound or composition as disclosed herein. The methods canfurther comprise administering a second compound or composition, suchas, for example, anticancer agents or anti-inflammatory agents.Additionally, the method can further comprise administering an effectiveamount of ionizing radiation to the subject.

Methods of killing a tumor cell are also provided herein. The methodscomprise contacting a tumor cell with an effective amount of a compoundor composition as disclosed herein. The methods can further includeadministering a second compound or composition (e.g., an anticanceragent or an anti-inflammatory agent) or administering an effectiveamount of ionizing radiation to the subject.

Also provided herein are methods of radiotherapy of tumors, comprisingcontacting the tumor with an effective amount of a compound orcomposition as disclosed herein and irradiating the tumor with aneffective amount of ionizing radiation.

Also disclosed are methods for treating oncological disorders in apatient. In one embodiment, an effective amount of one or more compoundsor compositions disclosed herein is administered to a patient having anoncological disorder and who is in need of treatment thereof. Thedisclosed methods can optionally include identifying a patient who is orcan be in need of treatment of an oncological disorder. The patient canbe a human or other mammal, such as a primate (monkey, chimpanzee, ape,etc.), dog, cat, cow, pig, or horse, or other animals having anoncological disorder. Oncological disorders include, but are not limitedto, cancer and/or tumors of the anus, bile duct, bladder, bone, bonemarrow, bowel (including colon and rectum), breast, eye, gall bladder,kidney, mouth, larynx, esophagus, stomach, testis, cervix, head, neck,ovary, lung, mesothelioma, neuroendocrine, penis, skin, spinal cord,thyroid, vagina, vulva, uterus, liver, muscle, pancreas, prostate, bloodcells (including lymphocytes and other immune system cells), and brain.Specific cancers contemplated for treatment include carcinomas,Karposi's sarcoma, melanoma, mesothelioma, soft tissue sarcoma,pancreatic cancer, lung cancer, leukemia (acute lymphoblastic, acutemyeloid, chronic lymphocytic, chronic myeloid, and other), and lymphoma(Hodgkin's and non-Hodgkin's), and multiple myeloma.

Administration

The disclosed compounds can be administered either sequentially orsimultaneously in separate or combined pharmaceutical formulations. Whenone or more of the disclosed compounds is used in combination with asecond therapeutic agent, the dose of each compound can be either thesame as or differ from that when the compound is used alone. Appropriatedoses will be readily appreciated by those skilled in the art.

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound as described herein meansintroducing the compound or a prodrug of the compound into the system ofthe animal in need of treatment. When a compound as described herein orprodrug thereof is provided in combination with one or more other activeagents (e.g., a cytotoxic agent, etc.), “administration” and itsvariants are each understood to include concurrent and sequentialintroduction of the compound or prodrug thereof and other agents.

In vivo application of the disclosed compounds, and compositionscontaining them, can be accomplished by any suitable method andtechnique presently or prospectively known to those skilled in the art.For example, the disclosed compounds can be formulated in aphysiologically- or pharmaceutically-acceptable form and administered byany suitable route known in the art including, for example, oral, nasal,rectal, topical, and parenteral routes of administration. As usedherein, the term parenteral includes subcutaneous, intradermal,intravenous, intramuscular, intraperitoneal, and intrastemaladministration, such as by injection. Administration of the disclosedcompounds or compositions can be a single administration, or atcontinuous or distinct intervals as can be readily determined by aperson skilled in the art.

The compounds disclosed herein, and compositions comprising them, canalso be administered utilizing liposome technology, slow releasecapsules, implantable pumps, and biodegradable containers. Thesedelivery methods can, advantageously, provide a uniform dosage over anextended period of time. The compounds can also be administered in theirsalt derivative forms or crystalline forms.

The compounds disclosed herein can be formulated according to knownmethods for preparing pharmaceutically acceptable compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin (1995)describes formulations that can be used in connection with the disclosedmethods. In general, the compounds disclosed herein can be formulatedsuch that an effective amount of the compound is combined with asuitable carrier in order to facilitate effective administration of thecompound. The compositions used can also be in a variety of forms. Theseinclude, for example, solid, semi-solid, and liquid dosage forms, suchas tablets, pills, powders, liquid solutions or suspension,suppositories, injectable and infusible solutions, and sprays. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically-acceptable carriers and diluents which areknown to those skilled in the art. Examples of carriers or diluents foruse with the compounds include ethanol, dimethyl sulfoxide, glycerol,alumina, starch, saline, and equivalent carriers and diluents. Toprovide for the administration of such dosages for the desiredtherapeutic treatment, compositions disclosed herein can advantageouslycomprise between about 0.1% and 99%, and especially, 1 and 15% by weightof the total of one or more of the subject compounds based on the weightof the total composition including carrier or diluent.

Formulations suitable for administration include, for example, aqueoussterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions, which can include suspending agents and thickening agents.The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions disclosed herein can include other agents conventional inthe art having regard to the type of formulation in question.

Compounds disclosed herein, and compositions comprising them, can bedelivered to a cell either through direct contact with the cell or via acarrier means. Carrier means for delivering compounds and compositionsto cells are known in the art and include, for example, encapsulatingthe composition in a liposome moiety. Another means for delivery ofcompounds and compositions disclosed herein to a cell comprisesattaching the compounds to a protein or nucleic acid that is targetedfor delivery to the target cell. U.S. Pat. No. 6,960,648 and U.S.Application Publication Nos. 2003/0032594 and 2002/0120100 discloseamino acid sequences that can be coupled to another composition and thatallows the composition to be translocated across biological membranes.U.S. Application Publiation No. 2002/0035243 also describes compositionsfor transporting biological moieties across cell membranes forintracellular delivery. Compounds can also be incorporated intopolymers, examples of which include poly (D-L lactide-co-glycolide)polymer for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL);chondroitin; chitin; and chitosan.

For the treatment of oncological disorders, the compounds disclosedherein can be administered to a patient in need of treatment incombination with other antitumor or anticancer substances and/or withradiation and/or photodynamic therapy and/or with surgical treatment toremove a tumor. These other substances or treatments can be given at thesame as or at different times from the compounds disclosed herein. Forexample, the compounds disclosed herein can be used in combination withmitotic inhibitors such as taxol or vinblastine, alkylating agents suchas cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracilor hydroxyurea, DNA intercalators such as adriamycin or bleomycin,topoisomerase inhibitors such as etoposide or camptothecin,antiangiogenic agents such as angiostatin, antiestrogens such astamoxifen, and/or other anti-cancer drugs or antibodies, such as, forexample, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN(Genentech, Inc.), respectively.

Many tumors and cancers have viral genome present in the tumor or cancercells. For example, Epstein-Barr Virus (EBV) is associated with a numberof mammalian malignancies. The compounds disclosed herein can also beused alone or in combination with anticancer or antiviral agents, suchas ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc., to treatpatients infected with a virus that can cause cellular transformationand/or to treat patients having a tumor or cancer that is associatedwith the presence of viral genome in the cells. The compounds disclosedherein can also be used in combination with viral based treatments ofoncologic disease. For example, the compounds can be used with mutantherpes simplex virus in the treatment of non-small cell lung cancer(Toyoizumi, et al., “Combined therapy with chemotherapeutic agents andherpes simplex virus type IICP34.5 mutant (HSV-1716) in human non-smallcell lung cancer,” Human Gene Therapy, 1999, 10(18):17).

Therapeutic application of compounds and/or compositions containing themcan be accomplished by any suitable therapeutic method and techniquepresently or prospectively known to those skilled in the art. Further,compounds and compositions disclosed herein have use as startingmaterials or intermediates for the preparation of other useful compoundsand compositions.

Compounds and compositions disclosed herein can be locally administeredat one or more anatomical sites, such as sites of unwanted cell growth(such as a tumor site or benign skin growth, e.g., injected or topicallyapplied to the tumor or skin growth), optionally in combination with apharmaceutically acceptable carrier such as an inert diluent. Compoundsand compositions disclosed herein can be systemically administered, suchas intravenously or orally, optionally in combination with apharmaceutically acceptable carrier such as an inert diluent, or anassimilable edible carrier for oral delivery. They can be enclosed inhard or soft shell gelatin capsules, can be compressed into tablets, orcan be incorporated directly with the food of the patient's diet. Fororal therapeutic administration, the active compound can be combinedwith one or more excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,aerosol sprays, and the like.

The tablets, troches, pills, capsules, and the like can also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring can be added. Whenthe unit dosage form is a capsule, it can contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials can be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules can be coatedwith gelatin, wax, shellac, or sugar and the like. A syrup or elixir cancontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound canbe incorporated into sustained-release preparations and devices.

Compounds and compositions disclosed herein, including pharmaceuticallyacceptable salts, hydrates, or analogs thereof, can be administeredintravenously, intramuscularly, or intraperitoneally by infusion orinjection. Solutions of the active agent or its salts can be prepared inwater, optionally mixed with a nontoxic surfactant. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient, which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid, and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating a compoundand/or agent disclosed herein in the required amount in the appropriatesolvent with various other ingredients enumerated above, as required,followed by filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, compounds and agents disclosed herein can beapplied in as a liquid or solid. However, it will generally be desirableto administer them topically to the skin as compositions, in combinationwith a dermatologically acceptable carrier, which can be a solid or aliquid. Compounds and agents and compositions disclosed herein can beapplied topically to a subject's skin to reduce the size (and caninclude complete removal) of malignant or benign growths, or to treat aninfection site. Compounds and agents disclosed herein can be applieddirectly to the growth or infection site. Preferably, the compounds andagents are applied to the growth or infection site in a formulation suchas an ointment, cream, lotion, solution, tincture, or the like. Drugdelivery systems for delivery of pharmacological substances to dermallesions can also be used, such as that described in U.S. Pat. No.5,167,649.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver a compound to the skin are disclosed inU.S. Pat. No. 4,608,392; U.S. Pat. No. 4,992,478; U.S. Pat. No.4,559,157; and U.S. Pat. No. 4,820,508.

Useful dosages of the compounds and agents and pharmaceuticalcompositions disclosed herein can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art; for example, see U.S. Pat. No. 4,938,949.

Also disclosed are pharmaceutical compositions that comprise a compounddisclosed herein in combination with a pharmaceutically acceptablecarrier. Pharmaceutical compositions adapted for oral, topical orparenteral administration, comprising an amount of a compound constitutea preferred aspect. The dose administered to a patient, particularly ahuman, should be sufficient to achieve a therapeutic response in thepatient over a reasonable time frame, without lethal toxicity, andpreferably causing no more than an acceptable level of side effects ormorbidity. One skilled in the art will recognize that dosage will dependupon a variety of factors including the condition (health) of thesubject, the body weight of the subject, kind of concurrent treatment,if any, frequency of treatment, therapeutic ratio, as well as theseverity and stage of the pathological condition.

For the treatment of oncological disorders, compounds and agents andcompositions disclosed herein can be administered to a patient in needof treatment prior to, subsequent to, or in combination with otherantitumor or anticancer agents or substances (e.g., chemotherapeuticagents, immunotherapeutic agents, radiotherapeutic agents, cytotoxicagents, etc.) and/or with radiation therapy and/or with surgicaltreatment to remove a tumor. For example, compounds and agents andcompositions disclosed herein can be used in methods of treating cancerwherein the patient is to be treated or is or has been treated withmitotic inhibitors such as taxol or vinblastine, alkylating agents suchas cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracilor hydroxyurea, DNA intercalators such as adriamycin or bleomycin,topoisomerase inhibitors such as etoposide or camptothecin,antiangiogenic agents such as angiostatin, antiestrogens such astamoxifen, and/or other anti-cancer drugs or antibodies, such as, forexample, GLEEVEC (Novartis Pharmaceuticals Corporation; East Hanover,N.J.) and HERCEPTIN (Genentech, Inc.; South San Francisco, Calif.),respectively. These other substances or radiation treatments can begiven at the same as or at different times from the compounds disclosedherein. Examples of other suitable chemotherapeutic agents include, butare not limited to, altretamine, bleomycin, bortezomib (VELCADE),busulphan, calcium folinate, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, crisantaspase, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel,doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gefitinib(IRESSA), gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib(GLEEVEC), irinotecan, liposomal doxorubicin, lomustine, melphalan,mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin,paclitaxel, pentostatin, procarbazine, raltitrexed, streptozocin,tegafur-uracil, temozolomide, thiotepa, tioguanine/thioguanine,topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine.In an exemplified embodiment, the chemotherapeutic agent is melphalan.Examples of suitable immunotherapeutic agents include, but are notlimited to, alemtuzumab, cetuximab (ERBITUX), gemtuzumab, iodine 131tositumomab, rituximab, trastuzamab (HERCEPTIN). Cytotoxic agentsinclude, for example, radioactive isotopes (e.g., I¹³¹, I¹²⁵, Y⁹⁰, P³²,etc.), and toxins of bacterial, fungal, plant, or animal origin (e.g.,ricin, botulinum toxin, anthrax toxin, aflatoxin, jellyfish venoms(e.g., box jellyfish, etc.) Also disclosed are methods for treating anoncological disorder comprising administering an effective amount of acompound and/or agent disclosed herein prior to, subsequent to, and/orin combination with administration of a chemotherapeutic agent, animmunotherapeutic agent, a radiotherapeutic agent, or radiotherapy.

Kits

Kits for practicing the methods described herein are further provided.By “kit” is intended any manufacture (e.g., a package or a container)comprising at least one reagent, e.g., anyone of the compounds describedin Table 1. The kit can be promoted, distributed, or sold as a unit forperforming the methods described herein. Additionally, the kits cancontain a package insert describing the kit and methods for its use. Anyor all of the kit reagents can be provided within containers thatprotect them from the external environment, such as in sealed containersor pouches.

To provide for the administration of such dosages for the desiredtherapeutic treatment, in some embodiments, pharmaceutical compositionsdisclosed herein can comprise between about 0.1% and 45%, andespecially, 1 and 15%, by weight of the total of one or more of thecompounds based on the weight of the total composition including carrieror diluents. Illustratively, dosage levels of the administered activeingredients can be: intravenous, 0.01 to about 20 mg/kg;intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about200 mg/kg, and preferably about 1 to 100 mg/kg; intranasal instillation,0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal(body) weight.

Also disclosed are kits that comprise a composition comprising acompound disclosed herein in one or more containers. The disclosed kitscan optionally include pharmaceutically acceptable carriers and/ordiluents. In one embodiment, a kit includes one or more othercomponents, adjuncts, or adjuvants as described herein. In anotherembodiment, a kit includes one or more anti-cancer agents, such as thoseagents described herein. In one embodiment, a kit includes instructionsor packaging materials that describe how to administer a compound orcomposition of the kit. Containers of the kit can be of any suitablematerial, e.g., glass, plastic, metal, etc., and of any suitable size,shape, or configuration. In one embodiment, a compound and/or agentdisclosed herein is provided in the kit as a solid, such as a tablet,pill, or powder form. In another embodiment, a compound and/or agentdisclosed herein is provided in the kit as a liquid or solution. In oneembodiment, the kit comprises an ampoule or syringe containing acompound and/or agent disclosed herein in liquid or solution form.

Method of Screening

Also disclosed herein are methods of identifying a putative anti-cancercompound comprising contacting an Aurora kinase with a target compoundand determining whether the compound binds the kinase in a DFG-outconfiguration, wherein a compound that binds the DFG-out configurationis identified as a putative anti-cancer compound.

EXAMPLES

The following examples are set forth below to illustrate the methods,compositions, and results according to the disclosed subject matter.These examples are not intended to be inclusive of all aspects of thesubject matter disclosed herein, but rather to illustrate representativemethods, compositions, and results. These examples are not intended toexclude equivalents and variations, which are apparent to one skilled inthe art.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric. There are numerous variations and combinations ofreaction conditions, e.g., component concentrations, temperatures,pressures, and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompositions are either available from commercial suppliers such asAcros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh,Pa.), or Sigma Aldrich (St. Louis, Mo.) or are prepared by methods knownto those skilled in the art following procedures set forth in referencessuch as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17(John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds,Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989);Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March'sAdvanced Organic Chemistry, (John Wiley and Sons, 4th Edition); andLarock's Comprehensive Organic Transformations (VCH Publishers Inc.,1989).

Standard techniques were used for molecular, genetic and biochemicalmethods. See, generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols inMolecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.; as well asGuthrie et al., Guide to Yeast Genetics and Molecular Biology, Methodsin Enzymology, Vol. 194, Academic Press, Inc., (1991), PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), McPherson et al., PCR Volume 1, Oxford UniversityPress, (1991), Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), and Gene Transferand Expression Protocols, pp. 109-128, ed. E. J. Murray, The HumanaPress Inc., Clifton, N.J.).

Reagents and compounds for biochemical and crystallization experimentswere purchased from Sigma-Aldrich and Hampton Research unless otherwiseindicated. The peptide substrate for activity assays was synthesized inthe Moffitt Cancer Center Proteomics Core, and the Aurora inhibitorVX680 was from LC laboratories. During protein purification, the proteinconcentration was determined using the Coomassie reagent from BioRadwith bovine serum albumin as a standard. The concentration ofcrystallization grade proteins was determined by A280 molar absorbanceusing a nanodrop ND-1000 spectrophotometer (Nanodrop Technologies).Non-linear regression analysis was performed using SigmaPlot (SystatSoftware).

Cloning and Expression.

The gene coding for the human Aurora A kinase domain comprising residues123-390 was synthesized by Geneart (Heidelberg, Germany) and subclonedinto a modified pET28 plasmid to provide fusion with cleavablehexahistidine-tagged maltose binding protein (MBP). The protein wasoverexpressed in E. coli Tuner (DE3) cells at 16° C.

Protein Purification.

The enzyme was purified by FPLC using Ni²⁺-affinity chromatography (GEHealthcare; Piscataway, N.J.) in 50 mM phosphate (pH 7.2) and 300 mMNaCl with a linear gradient of 10-250 mM imidazole, followed byovernight digestion with PreScission protease at 4° C. The protein wasexchanged into 50 mM MES (pH 6.5) and 1 mM DTF by rapid gel filtration,and the cleaved Aurora A construct and His6-MBP proteins were separatedusing a SP Sepharose Fast Flow ion exchange column (GE Healthcare),eluted with a 0-500 mM NaCl linear gradient. The pooled eluate was thenconcentrated and loaded onto a HiLoad Prep Grade 26/60 Superdex 200column (GE Healthcare) equilibrated with 50 mM HEPES (pH 7.4), 100 mMNaCl, and 1 mM DIT. The resulting eluate yielded crystallization-grademonomeric enzyme.

Isothermal Titration Calorimetry (ITC).

The binding of inhibitors to Aurora A kinase was analyzed with aMicroCal iTC200 titration calorimeter (GE Healthcare, Piscataway, N.J.).The protein was exchanged into 100 mM Na/K phosphate (pH 7.4) via PD-10columns (GE Healthcare Lifesciences; Piscataway, N.J.). A total of 18aliquots (2.2 μL each) of the protein solution (125 μM) were injectedinto 200 μL of the inhibitor solution (10 μM) at 25° C. The ITC cellmixture was constantly stirred at 1000 rpm and recorded for 120 secondsbetween injections. Heat generation due to dilution (blank) wasdetermined in a separate experiment by diluting protein into buffer. Thecorrected heat values were fitted using a nonlinear least squarecurve-fitting algorithm (Microcal Origin 7.0) to obtain thestoichiometry (n), binding constant (K_(a), K_(d)) and change inenthalpy of the enzyme-ligand interaction (ΔH). The ITC graphs are shownin the FIG. 20A through FIG. 20E.

Protein Crystallography.

Aurora A was exchanged into 50 mM phosphate buffer (pH 7.4) including 1mM DT via PD-10 columns and was concentrated to 20 mg mL⁻¹ using AmiconUltra-4 10K centrifugal devices (Millipore; Billerica, Mass.). Aurora Acrystals were grown using sitting drop vapor diffusion at 18° C. from a1:1 volume ratio of Aurora A and reservoir solution (200 mM sodiumtartrate/20% polyethylene glycol 3350 for the DFG-in inhibitors or 10%Tacsimate/20% polyethylene glycol 3350 for the DFG-out inhibitors) witha final inhibitor concentration of 1 mM. Crystals appeared after twodays and were allowed to grow for another 2 days. For data collection,crystals were harvested in a cryoprotectant mixture consisting of therespective reservoir composition including 1 mM inhibitor, 50 mMphosphate pH (7.4) and 25% (v/v) ethylene glycol. Diffraction data wererecorded at −180° C. in the Moffitt Cancer Center Chemical Biology Coreusing CuKα X-rays generated by a Rigaku Micro-Max 007-HF X-raygenerator, focused by mirror optics and equipped with a Rigaku CCDSaturn 944 system on single crystals frozen in liquid N₂. Data werereduced with XDS (Kabsch, (1993) Automatic processing of rotationdiffraction data from crystals of initially unknown symmetry and cellconstants, J Appl Crystallogr 26:795-800). The ‘DFG-in’ crystalsbelonged to the P6₁22 space group, containing one monomer in theasymmetric unit. The ‘DFG-out’ crystals belonged to space group P3₂ withtwo monomers in the asymmetric unit. The structures were determined bymolecular replacement using MOLREP (CCP4) (1994) The CCP4 suite:programs for protein crystallography, Acta Crystallogr D BiolCrystallogr 50:760-763) and PDB entry 3FDN as the search model. PHENIX(Adams et al., (2010) PHENIX: a comprehensive Python-based system formacromolecular structure solution, Acta Crystallogr D Biol Crystallogr66:213-221) was employed for refinement (minimization and simulatedannealing), and model building was performed using Coot (Emsley et al.,(2004) Coot: model-building tools for molecular graphics, ActaCrystallogr D Biol Crystallogr 60:2126-2132). Figures were drawn withPyMol (SchrOdinger, LLC). CDK2 was co-crystallized with compounds 1 and7 using previously established conditions (Betzi et al., (2011)Discovery of a potential allosteric ligand binding site in CDK2, ACSChem Biol 6:492-501) and the structures were determined accordingly.Data and refinement statistics are shown in Table A, along with theFo-Fc electron density maps from refinement cycles omitting therespective ligand (FIG. 23A through FIG. 25B).

TABLE A Summary of data collection and structure refinement^(a).Structure 1 3 4 5 (PDB ID) (3UO5) (3UO4) (3UOD) (3UP2) Data CollectionSpace group P6₁22 P6₁22 P6₁22 P6₁22 Unit cell dimensions (Å) a = 81.79 a= 83.08 a = 81.98 a = 82.44 b = 81.79 b = 83.08 b = 81.98 b = 82.44 c =173.77 c = 172.76 c = 173.81 c = 173.14 Resolution range 20-2.70 20-2.4520-2.50 20-2.30 (2.75-2.70) (2.50-2.45) (2.60-2.50) (2.40-2.30) Uniquereflections 9540 (473)  13594 (773)  12584 (1353)  16126 (1860) Completeness (%) 94.9 (96.7) 99.6 (99.9) 99.7 (99.9) 99.7 (99.9) I/σI36.9 (7.3)  60.9 (9.1)  51.8 (7.8)  54.4 (10.7) R_(merge) ^(b) (%) 14.1(31.6)  3.4 (20.7)  4.4 (22.9)  3.1 (15.8) Structure refinement Proteinatoms 2195 2188 2188 2188 Average B-factor (Å²) 42 58 52 50 Ligand atoms23 29 27 28 Average B-factor (Å²) 29 43 39 38 Solvent molecules 36 49 4598 Average B-factor (Å²) 33 46 44 46 r.m.s.d.^(c) bonds (Å) 0.014 0.0040.009 0.010 r.m.s.d. angles (°) 1.2 1.0 1.2 1.2 R_(cryst) ^(d) (%) 21.522.0 20.6 20.8 R_(free) ^(e) (%) 27.4 25.7 26.2 24.9 R_(free) reflectionset size  393 (4.5%)  612 (4.5%)  567 (4.5%)  775 (4.5%) Coordinateerror (Å) 0.40 0.27 0.22 0.17 Structure 6 7 8 9 (PDB ID) (3UNZ) (3UO6)(3UOH) (3UOJ) Data Collection Space group P3₂ P3₂ P3₂ P3₂ Unit celldimensions (Å) a = 85.78 a = 85.71 a = 85.75 a = 85.71 b = 85.78 b =85.71 b = 85.75 b = 85.71 c = 76.56 c = 76.66 c = 76.94 c = 77.08Resolution range 20-2.80 20-2.80 20-2.80 20-2.90 (2.90-2.80) (2.90-2.80)(2.90-2.80) (3.00-2.90) Unique reflections 15425 (1514)  15518 (1509) 15512 (1541)  13920 (1330)  Completeness (%) 99.4 (99.4) 99.0 (99.7)99.5 (99.9) 99.1 (99.0) I/σI 27.6 (4.6)  31.8 (4.7)  30.9 (5.3)  25.5(4.5)  R_(merge) ^(b) (%)  7.8 (39.7)  6.8 (37.4)  6.2 (32.2)  6.5(41.1) Structure refinement Protein atoms 4318 4340 4340 4318 AverageB-factor (Å²) 58 69 64 70 Ligand atoms 48 48 48 50 Average B-factor (Å²)39 42 42 43 Solvent molecules 38 21 34 28 Average B-factor (Å²) 44 46 4449 r.m.s.d.^(d) bonds (Å) 0.007 0.013 0.009 0.013 r.m.s.d. angles (°)1.3 1.5 1.2 1.9 R_(cryst) ^(c) (%) 23.1 22.9 23.0 22.7 R_(free) ^(f) (%)27.7 27.6 28.8 27.4 R_(free) reflection set size  695 (4.5%)  614 (4.0%) 699 (4.5%)  557 (4.0%) Coordinate error (Å) 0.42 0.41 0.42 0.48Structure 10 11 CDK2-1 CDK2-7 (PDB ID) (3UOK) (3UOL) (3UNJ) (3UNK) DataCollection Space group P3₂ P3₂ P2₁2₁2₁ P2₁2₁2₁ Unit cell dimensions (Å)a = 85.74 a = 85.75 a = 53.22 a = 52.95 b = 85.74 b = 85.75 b = 71.96 b= 72.02 c = 76.69 c = 76.72 c = 72.65 c = 72.37 Resolution range 20-2.9520-2.40 20-1.90 20-2.10 (3.00-2.95) (2.50-2.40) (2.00-1.90) (2.20-2.10)Unique reflections 13174 (598)  24248 (2768)  22465 (3133)  16682(2127)  Completeness (%) 99.5 (99.8) 98.4 (98.0) 99.4 (99.2)  100 (99.8)I/σI 22.4 (3.8)  30.2 (7.5)  18.5 (7.5)  20.7 (6.9)  R_(merge) ^(b) (%) 7.7 (47.4)  4.4 (23.9)  5.9 (23.2)  7.5 (23.8) Structure refinementProtein atoms 4358 4320 2366 2371 Average B-factor (Å²) 57 58 27 28Ligand atoms 50 52 23 24 Average B-factor (Å²) 36 43 21 25 Solventmolecules 17 124 214 153 Average B-factor (Å²) 37 50 31 27 r.m.s.d.^(d)bonds (Å) 0.010 0.012 0.010 0.010 r.m.s.d. angles (°) 1.5 1.5 1.4 1.3R_(cryst) ^(e) (%) 22.3 23.6 19.9 18.0 R_(free) ^(f) (%) 27.6 27.9 26.123.4 R_(free) reflection set size  525 (4.0%) 1067 (4.4%) 1124 (5.0%) 835 (5.0%) Coordinate error (Å) 0.44 0.40 0.25 0.21 ^(a)The structurewith compound 2 has been deposited as PDB code 3UP7 (Lawrence et al,submitted). ^(b)R_(merge) = quality of amplitudes (F) in the scaled dataset, Diederichs & Karplus (1997), Nature Struct. Biol. 4, 269-275.^(c)r.m.s.d. = root mean square deviation from ideal values.^(d)R_(cryst) = 100 × Σ|F_(obs) − F_(model)|/F_(obs) where F_(obs) andF_(model) are observed and calculated structure factor amplitudes,respectively. ^(e)R_(free) is R_(cryst) calculated for randomly chosenunique reflections, which were excluded from the refinement.

Accession Codes.

The atomic coordinates and structure factors for Aurora A in complexwith compounds 1 and 3-11, and CDK2 in complex with compounds 1 and 7,have been deposited under accession numbers 3UO5, 3UO4, 3UOD, 3UP2,3UNZ, 3UO6, 3UOH, 3UOJ, 3UOK, 3UOL, 3UNJ and 3UNK, respectively. Thecrystal structure of Aurora A with compound 2 has been deposited undercode 3UP7.

Example 1 SAR Studies

SAR studies were initiated while attempts were being made toco-crystallize compound 1 with Aurora A. Focused library synthesis basedon 1 (FIG. 1A through FIG. 1C) was first undertaken varying 4 points ofmolecular diversity (R¹, R², R³ and R⁴, see FIG. 1C) by systematicallyreplacing or introducing the functional groups in the A and B-rings.Replacement of the B ring para-carboxylic acid in 1 by hydrogen or amorpholino as in 3b or 3c resulted in 13- and 10-fold loss of inhibitoryactivity, respectively (Entries 3 and 4, Table 1). Replacing bothcarboxylic acid moieties in 1 with hydrogen as in 3f (Entry 7, Table 1)resulted in 70-fold loss of potency. In addition, replacement of bothcarboxylic acid moieties by corresponding methyl esters as shown in 3e(Entry 6, Table 1) resulted in over 4000-fold loss of potency. However,compound 3g (Entry 8, Table 1) with carboxyamide in ortho- andpara-positions of the A and B rings respectively was only 6-fold lesspotent than the compound 1. The SAR data showed the positions of bothcarboxylic acid moieties in A and B rings are critical for activity.Moving the B ring para-carboxylic acid moiety to the ortho-position, asin 3d, demonstrated 5000-fold loss of potency (Entry 5, Table 1,IC₅₀=31,300 nM). The compound 3q (Entry 18, Table 1, IC₅₀=18.3 nM) withmeta-carboxylic acid in the B ring had a 3-fold loss of in vitropotency. Furthermore, moving the A ring carboxylic acid moiety incompound 1 from ortho- to para-position, as in 13i (Entry 10, Table 1),resulted in 42 fold loss of potency. Replacement of the B ringpara-carboxylic acid by ortho-amide (compound 3a) resulted in >than1000-fold loss of Aurora A inhibitory activity (Entries 2, Table 1).These observations suggested that the ortho-position of the A ring andpara-position of the B ring are associated with enzymatic activity andfocused library synthesis.

The X-ray co-crystal structure of 1 bound to Aurora A supports the abovefindings (FIG. 2A through FIG. 2E). Analysis of this structure shows thepara-carboxylic acid group of the B ring forming key H-bond interactionswith the solvent exposed residues Arg137 and Arg220 (FIG. 2A and FIG.2B). Compound 1 is a type I kinase inhibitor that targets the ATPbinding site (FIG. 2C). The pyrimidine scaffold and the amine moiety ofthe B-ring establish H-bonding with the hinge region (residues 211-213,FIG. 2A and FIG. 22B). Analysis of ADP/ATP bound to Aurora A indicatedhighly conserved residues Lys162, Asp274, and Glu181 undergoingelectrostatic interactions with ADP phosphate moieties in the activesite (FIG. 2D). Compound 1 bound to Aurora A shows these key residues inthe active site are now in contact with the ortho-COOH moiety of theA-ring (FIG. 2B). The ortho-carboxylic acid of the A-ring in compound 1is in the vicinity (3.5 Å) to form an electrostatic interaction withLys162 (Figure FIG. 2B). The carboxylic acid moiety of the Asp-274 ofthe DFG motif (FIG. 2A and FIG. 2B) is also in close distance (3.2 Å)with Lys162 and the ortho-COOH moiety of compound 1 to form an H-bond.This key H-bonding network contributes to high in-vitro potency of thecompound 1. This is consistent with the fact that compound 3f (Entry 7,Table 1), where both A and B-rings contain unsubstituted phenyl, was70-fold less active than parent compound 1, and the dimethyl ester 3elost the in-vitro inhibitory activity (IC₅₀ 24.6 μM, Entry 6, Table 1).These observations further confirm that the key interactions observedfrom the X-ray structure contribute to inhibitory activity (FIG. 2B).The loss of inhibitory activity observed with 3d (Entry 5, Table 1) inthe SAR studies is consistent with the X-ray structure of compound 1bound to Aurora A where ortho-substituted B ring causes steric clashwith the main chain residues Ala213 and Pro214 (FIG. 2A and FIG. 2B).However, the activity of compound 3q is retained with carboxylic acid inthe meta-position, and improved when fluorine is added as in 3r (Entries18 and 19 table 1). The meta-COOH is able to interact with Arg220 andArg137.

Several X-ray structures were obtained to detail the binding modes ofthis class of compounds with Aurora A (FIG. 12A through FIG. 12D).Substitution of both carboxylic acid groups in compound 1 by primaryamides (FIG. 12B) reduced the inhibitory activity 6-fold (Entry 8, 3g,Table 1). The compound 3i (FIG. 12A), where the A-ring ortho-carboxylicacid is moved to the para-position, was 42-fold less potent than parentmolecule (Table 1, Entry 10, IC₅₀=256 nM). Not to be bound by theory,this decrease in inhibitory activity may be due to lack of key bindinginteractions of the para-carboxyl or para-carboxamide groups in theA-ring with the Lys162 and Asp274 active site residues (FIG. 12A andFIG. 12B, compounds 3i and 3g bound to Aurora A). In contrast, removalof the ortho-carboxylic group, as in 3h (Entry 9, Table 1), retainsactivity. The co-crystal structure of compound 3i (FIG. 12A through FIG.12D) does not explain the differences in activities of compounds 3h and3i. Compound 14 (FIG. 12C), where A-ring has a CF₃ in the meta-position,is a weaker binder (IC₅₀=371 nM,) and has the trifluoromethyl grouppositioned in the region that binds the diposphate group of ADP (FIG.2E). Thus, the presence of a hydrophobic group close to Asn261 andGlu260 does not contribute to binding affinity of Aurora A. Overallmeta-substituents in the A-ring did not improve the activity.

The surprising observation that replacement of the ortho-carboxylic acidof the A ring in compound 1 with hydrogen as in 3h did not result ingreat loss of potency prompted the further SAR analysis at thisposition. Therefore, library 6 (FIG. 5C) was synthesized to detail thebinding modes of Aurora A to bisanilinopyrimidines with different groupsin the ortho-position of the A ring. To this end, analogs of compound 1were synthesized by replacing the carboxylic acid in the ortho-positionof the A ring by fluoro, chloro, bromo, iodo, trifluoromethyl,trifluoromethoxy, methoxy, cyano, and phenyl as ortho-substituents(6a-6q, FIG. 5C). Compounds with halogens (F, Cl, Br) at theortho-position of the A ring and para-COOH moiety in the B ring(compounds 31 (Table 1) 6a and 6i (Table 2)) respectively improvedpotency by 1.5 to more than 3-fold, whereas compounds with bulkyhalogenated groups such as OCF₃ and CF₃ (Entries 27, 31 compounds 6d and6h respectively in Table 2) were much less active at inhibiting AuroraA. Compound 6q (Entry 40, Table 2) with an ortho-phenyl group was 24fold less potent than the compound 1, further indicating bulky groupsare not tolerated in this region (Entry 40, Table 2). Compound 6n(IC₅₀=43 nM, Entry 37, Table 2), that possesses an ortho-CN group, ismore than 17-fold less active than 31. The in vitro activity of 31(IC₅₀=2.5±0.3 nM, Entry 13, Table 1) with ortho-Cl and a para-COOH in Bring was further improved when R¹ is fluorine in compound 3o(IC₅₀=0.8±0.16 nM, Entry 16, Table 1). The para-COOH moiety in the Bring aided in maintaining the Aurora A inhibitory activity in library 6(FIG. 5C). The removal of COOH moiety resulted in loss of in vitropotency as observed with 6b, 6, 6g, and 61 (Entries 25, 29, 30 and 35,Table 2). The loss of in vitro inhibitory activity in compounds lackinga para-COOH moiety was a general trend observed with libraries 3 and 6and highlights the importance of H-bond interactions associated withArg137 and Arg220. Switching the position of B ring COOH moiety frompara- to meta-position as in compound 6o (Entry 38, Table 2) reduced theinhibitory activity compared to 3o (Entry 16, Table 1). Modification ofB ring para-COOH to para-CONH₂ in compound 6p reduced the in vitroactivity by 5-fold (Entry 39, Table 2). Compounds 6r and 6s (Entries 41and 42, Table 2) with N-alkylated moieties retained the activity;N-methyl derivative was highly potent (IC₅₀=8.5±1.2 nM) while theN-ethyl derivative was less active (IC₅₀=50.2±2.7 nM). Compound 6t(Entry 43, Table 2), with meta-COOH and fluorine as R¹, was 4-fold morepotent as compared to compound 6o, where R¹ is hydrogen. Overall, theSAR indicated fluorine or chlorine as R¹ in this series is beneficialand improves in vitro and in vivo Aurora A activity (see Table 3).

Alignment of the Aurora A-ADP complex with compound 1 indicated R¹ andR² in compound 1 as potential sites for synthetic modifications (FIG.2A, FIG. 2D, and FIG. 2E). The R¹ position (FIG. 1B) of the pyrimidinering is in close proximity (˜4 Å) to the gatekeeper residue Leu210 andR² is close to Glu211 (˜3.5 Å) (FIG. 2A and FIG. 2B). To exploit thisnarrow space, small groups such as methyl (Entry 11, 3j, Table 1), amine(Entry 22, 4a, Table 1), chlorine (Entries 20 and 21 3s and 3trespectively, Table 1), and fluorine (Entries 15 and 17. 3n-3p) werefirst introduced as R¹. The methyl and NH₂ derivatives, compounds 3j and4a respectively (Entries 11 and 22, Table 1), did not contribute toincreased activity most probably due to the steric effect of thesegroups unable to establish desired interactions. In contrast, compoundswith fluorine 3n, 3o (Entries 15-16, Table 1) and chlorine 3t (Entry 21,Table 1), were among the most potent inhibitors with IC₅₀ values between0.8-4 nM (Entries 15, 16 and 21, Table 1). It is likely that fluorineand chlorine as R¹ (FIG. 2B) undergo van der Waals interactions with theside chains of the hydrophobic pocket of Ala160, Leu194 and Leu210 (FIG.12D) as observed in the structures of fluoro-bisanilinopyrimidineinhibitors from Genentech. Substitution of the R² position (FIG. 1B) ofthe pyrimidine moiety with methyl, chloro, and amine, as shown incompounds 3k, 4c and 4b (Entries 12, 20 and 23 respectively, Table 1),decreased inhibitory activity, possibly due to steric clash with thebackbone carbonyl of Glu211. By contrast, as mentioned above, syntheticmodifications at R³ (FIG. 2A and FIG. 2B) are largely tolerated sincethe region around the DFG is less confined than that opposite hingeregion.

Aurora inhibitors that are cell permeable and able to inhibit Aurora Akinase in intact cells were next obtained. The potent compounds 3l, 3n,3o, 3t, 6a, 6j, 6k and 6r that showed low nanomolar activities in theenzymatic assay however showed poor aqueous solubility and poor activityin intact cells. Introduction of water-solubilizing groups at thepara-position of the B-ring was explored to improve both the solubilityand cell permeability. Therefore, substitution of the para-carboxylicacid in the B-ring with groups that contain a variety of neutral polarmoieties (FIG. 7A through FIG. 7B) were employed to exploit H-bondinteractions with Arg137 and Arg220. Direct replacement of the para-COOHby the carboxylic acid isostere tetrazole moiety provided compounds13a-13d that retained the in vitro potency similar to 3l (Entries 59, 60and 61, Table 3 and FIG. 8) with good aqueous solubility (49 μg/ml inDMEM buffer at pH 7.4). An X-ray structure of compound 13a (FIG. 8)bound to Aurora A was obtained, and compound 13a adopted a bindingconformation similar to compound 3l. The primary aim of the B-ringmodifications described in FIG. 7A, FIG. 7B and FIG. 8 was to obtain acompound with chlorine at the ortho-position of the A-ring and awater-solubilizing moiety at the para-position of the B-ring.

TABLE 1 Synthetic Modifications, structure activity relationship studiesand in-vitro activities of bisanilinipyrimidine libraries 3 and 4against Aurora A.

Cmpd In Vitro ID activity (IC₅₀) Entry # R¹ R² R³ R⁴ (DiscoverX)  1 1  HH ortho- para- 6.1 ± 1.0 nM COOH COOH  2 3a H H ortho- ortho- 9.0 ± 6.8μM COOH CONH₂  3 3b H H ortho- H 79.4 ± 18 nM   COOH  4 3c H H ortho-para- 57.6 ± 5.4 nM  COOH morpholine  5 3d H H ortho- ortho- 31.3 ± 5.9μM  COOH COOH  6 3e H H ortho- para- 24.6 ± 6.0 μM  COOMe COOMe  7 3f HH H H 423 ± 66 nM   8 3g H H ortho- para- 38.2 ± 8.8 nM  CONH₂  9 3h H HH para-  10 ± 1.6 nM COOH 10 3i H H para- para- 256 ± 38 nM  COOH COOH11 3j CH₃ H ortho- para- 281 ± 59 nM  COOH COOH 12 3k H CH₃ ortho-para- >50 μM COOH COOH 13 3l H H ortho-Cl para- 2.5 ± 0.3 nM COOH 14 3mF H ortho- All H 11.3 ± 1.7 nM  COOH 15 3n F H ortho- para- 3.9 ± 0.5 nMCOOH COOH 16 3o F H ortho-Cl para-  0.8 ± 0.16 nM COOH 17 3p F Hortho-Cl H 19.9 ± 2.2 nM  18 3q H H ortho- meta- 18.3 ± 3.4 nM  COOHCOOH 19 3r F H ortho- meta- 5.1 ± 1.1 nM COOH COOH 20 4c Cl Cl ortho-para-  1.49 ± 0.196 μM COOH COOH 21 4d Cl H ortho- para- 3.17 ± 0.51 nMCOOH COOH 22 4a NH₂ H ortho- para- 376 ± 64 nM  COOH COOH 23 4b H NH₂ortho- para- >50 μM COOH COOH

TABLE 2 Structure activity relationship studies and in-vitro inhibitoryactivities of library 6 against Aurora A.

Cmpd ID IC₅₀ (nM) Entry # R¹ R² R³ R⁴ R⁵ (DiscoverX) 24 6a H H ortho-Fpara-COOH H 3.7 ± 0.7 nM 25 6b H H ortho-CF₃ All H H 1773 ± 142 nM  266c H H 2-Cl—4-F para-COOH H 2.0 ± 0.2 nM 27 6d H H ortho-OCF₃ para-COOHH  28 ± 4.8 nM 28 6e H H ortho-OMe para-COOH H 4.0 ± 0.2 nM 29 6f H Hortho-OMe All H H 46.6 ± 8.4 nM  30 6g H H ortho-CN All H H  560 ± 70.3nM 31 6h H H ortho-CF₃ para-COOH H  35 ± 4.1 nM 31 6i H H ortho-Brpara-COOH H 2.1 ± 0.4 nM 33 6j H H ortho-Cl para-CH₂—COOH H 3.3 ± 1.5 nM34 6k H H Ortho-Cl para-COOH, meta-OH H 6.6 ± 0.6 nM 35 6l H H Ortho-FAll H H  284 ± 11.3 nM 36 6m H H Ortho-I para-COOH H  35 ± 3.3 nM 37 6nH H Ortho-CN para-COOH H 43 ± 58 nM 38 6o H H Ortho-Cl meta-COOH H 18.7± 1.5 nM  39 6p H H ortho-Cl para-CONH₂ H 30.2 ± 1.4 nM  40 6q H Hortho-phenyl para-COOH H 149 ± 23 nM  41 6r H H ortho-Cl para-COOH CH₃8.5 ± 1.2 nM 42 6s H H ortho-Cl para-COOH CH₃—CH₂ 50.2 ± 2.7 nM  43 6t FH ortho-Cl meta-COOH H 4.5 ± 1.1 nM

TABLE 3 In-vivo and in vitro Aurora A activities ofbisaniloinopyrimidines with water solubilizing moieties In-vivo IC₅₀(μM) Compound In-vitro [Aurora A inhibitory activity Entry ID IC₅₀ (nM)in MDA-MB-468 at 2h] 44

 216 ± 10.3 1-10 45

28.1 ± 5.5  1-10 46

 18 ± 2.8 1-10 47

44.9 ± 4.7  >10 48

71.2 ± 9.0  >10 49

 316 ± 44.2 1-10 50

116.5 ± 10.6  >10 51

 27 ± 7.6 <1  52

30.2 ± 1.4  <1  53

 253 ± 41.6 <1  54

21.4 ± 2.5  <1  55

23.2 ± 1.6  <1  56

12.3 ± 1.1  <1  57

14.4 ± 1.7  <1  58

21.2 ± 2.2  >10 59

 3.1 ± 0.16 >10 60

 2.9 ± 0.30 >10 61

17.3 ± 2.3  >10 62

 3.0 ± 0.53 1-10

TABLE 4 IC₅₀ data

IC₅₀ (μM) Compound Cmpd (in vitro) IC₅₀ (nM) ID # R¹ R² R³ R⁴ LDH/PK(DiscoverX) RK2-014 14 H H meta-CF₃ para-COOH 3.94 186.5 ± 26  RK2-017-01 15 H H meta-CF₃ para-CONH₂ 0.226 ± 0.046 371.5 ± 56   RK2-03716 H H meta-CF₃ meta-isobutyramide 1.4  1.43 ± 0.3 μM RK2-025 17 H Hmeta-CF₃ meta-CF₃ 10  23.7 ± 7.4 μM RK2-015-03 18 H H meta-CF₃ortho-COOH 64  25.7 ± 7.2 μM RK2-017-02 19 H H meta-CF₃ meta-CONH₂ 3.6 1.26 ± 0.2 μM RK2-053 20 H H meta-CF₃ meta-acetamide 0.91   3.6 ± 0.9μM RK2-015-02 21 H H meta-CF₃ meta-COOH 9.32  16.2 ± 7.3 μM RK2-056 22 HH meta-CF₃ meta-butyramide >15 — RK2-046-02 23 H H meta-CF₃meta-propionamide ?  15.4 ± 6.7 μM RK2-013 24 H H meta-CF₃meta-cyclopropane carboxamide ? >50 μM RK2-015-01 25 H H meta-CF₃ All H14.1 — RK2-046-01 26 H H meta-CF₃ meta-^(t)butylcarboxyamide 7.46  8.87± 2.9 μM RK2-044 27 H H meta-CF₃ meta-cyclopentylcarboxamide 6.25  23.0± 3.0 μM RK2-052 28 H H meta-CF₃ meta-isobutylcarboxamide >150  21.6 ±1.0 μM RK2-043 29 H H meta-CF₃ meta-(4-chlorobenzyl)carboxamide 10.2 >50μM RK2-049 30 H H meta-CF₃ meta-benzylcarboxamide 8.5 >50 μM YL5-048 31H Me₂N H All H >15 >50 μM YL5-050 32 H Me₂N ortho-COOH All H >15 >50 μMYL5-068 33 NH₂ H ortho-COOH para-COOC₂H₅ 0.26  1.49 ± 0.49 μM YL5-146-534 H H ortho-Cl para-OCH₃ YL5-080 35 Me H ortho-COOH para-COOH 0.145

Example 2 Effects of Aurora a Inhibitors on the Phosphorylation of theAurora a Substrate, Histone H3, in Breast Cancer Cells

The synthetic peptide LRRASLG served as a substrate for Aurora A.Formation of ADP from ATP was quantified using a coupled enzyme assay(DiscoverX, Fremont, Calif.) in which a fluorescent resorufin dye isgenerated from the interaction of ADP with hydrogen peroxide and10-acetyl-3,7-dihydroxy-phenoxazine (excitation and emission wavelengthsof 540 and 590 nm, respectively). Reactions were carried out at roomtemperature in 15 mM HEPES buffer (pH 7.4) containing 20 mM NaCl, 1 mMEGTA, 0.02% Tween 20, 10 mM MgCl₂, 5% (v/v) DMSO, and 2.3 nM Aurora A.Inhibitor was added to the mixture, and the reaction was initiated bythe addition of 75 μM ATP and 2 mM peptide substrate. All kinetic assayswere performed in 384-well plates using a Wallac Envision 2102 platereader (Perkin Elmer). IC₅₀ values for were obtained by fitting the datato equation (1),

$\begin{matrix}{A = \frac{1}{1 + \left( \frac{\lbrack I\rbrack}{{IC}_{50}} \right)^{n}}} & (1)\end{matrix}$

where A is the remaining activity, [I] is the concentration of theinhibitor, and n is the Hill slope coefficient. CDK2 IC₅₀ values weremeasured using the same assay as described (Betzi et al., (2011)Discovery of a potential allosteric ligand binding site in CDK2, ACSChem Biol 6:492-501). The dose-response curves are shown in thesupplemental material (FIG. 19).

The ability of the most potent Aurora A kinase inhibitors in vitro toblock the phosphorylation of serine 10 on Histone H3 was investigated, awell established Aurora A substrate. To this end, MDA-MB-468 breastcancer cells were treated with the inhibitors at various concentrationsfor 2 hours and processed for immunoblotting.

Specifically, MDA-MB-468 cells (American Type Culture Collection) weremaintained in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetusbovine serum (FBS) (Invitrogen, US) at 37° C., 5% CO₂. The cells wereplated in 6 cm dishes at a density of 2×10⁵ cells/dish. Cells were thentreated with the compounds (0-10 μM); DMSO was used as a negativecontrol and 0.5 μM VX-680 (Tozasertib) (Selleck Chemical LLC) was usedas a positive control. Cells were harvested after 2 h of treatment andprocessed for SDS-PAGE and western blotting as described previously(Gizatullin et al., “The Aurora kinase inhibitor VX-680 inducesendoreduplication and apoptosis preferentially in cells with compromisedp53-dependent postmitotic checkpoint function” Cancer Res. (2006) 66(15):7668-77). After electro-transfer onto nitrocellulose membranes, themembranes were blocked at room temperature for 1 h with TBS containing5% (w/v) milk and then washed with a mixture of TBS containing 0.2%Tween 20 (Sigma). The membranes were then gently shaken at 4° C.overnight with anti-phospho histone H₃ (Ser 10) antibody (9701, CellSignaling), and anti-GAPDH monoclonal antibody (E10086CF, Covance)diluted in TBS containing 5% BSA. The membranes were then incubated withHRP conjugated anti-rabbit or anti-mouse IgG antibody (JacksonImmunoResearch Lab.) at room temperature for 1 h followed by washingwith Tween 20-PBS. The membranes were washed again with PBS anddeveloped with the ECL system (PerkinElmer) as described previously(Berndt et al., “The Akt activation inhibitor TCN-P inhibits Aktphosphorylation by binding to the PH domain of Akt and blocking itsrecruitment to the plasma membrane.” Cell Death Difer. (2010) 17(11):1795-804).

FIG. 13A through FIG. 13C shows that compound 1, which contains acarboxylic acid moiety on each of the A-ring and the B-ring, as well asone of the most potent compounds 3l (in vitro IC₅₀=2.52±0.3 nM), wherethe A-ring carboxylate was replaced by a chlorine but still contains acarboxylate on the B-ring, had little effect on Histone H3phosphorylation levels in MDA-MB-468 cells. Similarly, 3o (in vitroIC₅₀=0.8±0.16 nM), which in addition to a carboxylate on the B-ring anda chlorine on the A-ring also contains a fluorine on the pyrimidinering, also had little effect on Histone H3 phosphorylation levels. Incontrast, 3p (in vitro IC₅₀=19.9±2.2 nM), which lacks both carboxylates,showed improved ability to inhibit Histone H3 phosphorylation. Theability of these compounds to inhibit Aurora A activity in intact cellswas determined by varying the substituents on the B-ring. FIG. 13Athrough FIG. 13C show that replacement of the B-ring carboxylate withtetrazole such as in 13a, 13b, 13c, and 13d did not improve activity. Incontrast, in compounds with a chlorine on the A-ring, substituting theB-ring carboxylate with a carboxamide (6p, Table 3), sulfonamide (9i,Table 3) or morpholino (9h, Table 3) greatly improved their activity andresulted in suppression of Histone H3 phosphorylation withconcentrations as low as 1 μM. Similarly, compounds with ortho-chlorineon the A-ring and fluorine as R¹ on the pyrimidine ring, substitutionsof the carboxylic acid moiety with carboxamide (9m, Table 3), morpholino(9j, Table 3), methylene morpholino (9k, Table 3), and sulfonamide (9n,Table 3), also greatly improved the ability to suppress Histone H3phosphorylation. Other modifications resulted in mediocre to goodimprovements, and these included analogues that occupiedthepara-position with NHCOCH₃ (9e, Table 3), SO₂CH₃ (9d, Table 3),CONHC₂H₄OCH₃ (9c, Table 1), CONHC₂H₄OH (9m, Table 3),CONHC₂H₄-morpholino (9a, Table 3), para-COOH and meta-OCH₃ (9g, Table 3)and CONHC₂H₄N(CH₃)₂ (9b, Table 3). Taken together, these resultsdemonstrate that in vitro Aurora A inhibitors that contain carboxylicgroups are inactive in intact cells but that those where the B-ringcarboxylic moiety was replaced by carboxamide, sulfonamide, ormorpholino groups are highly potent at inhibiting in intact cells thephosphorylation of the Aurora A kinase substrate.

Example 3 Selection of a Chemical Scaffold Suitable to Probe the DFGMotif of Aurora A

Compound 2 is a potent inhibitor of Aurora A with an IC₅₀ value of 6.1nM in vitro. The co-crystal structure confirmed that compound 2 is atypical Type I inhibitor which binds to the hinge region (Ala213)without perturbing the DFG-in state (Asp274-Phe275-Gly276) (FIG. 14Athrough FIG. 14I, Table 5). The unusually high potency of the hitcompound, the feasibility of focused library synthesis of thebisanilinopyrimidine scaffold, and the availability of robustco-crystallization conditions allowed the DFG region of Aurora A to betargeted for the design of DFG-out inhibitors. The ortho-position of theA-ring, as it is appropriately positioned for the introduction offunctional groups to target the DFG, was determined. Synthesizedanalogues were characterized by enzyme kinetics, isothermal titrationcalorimetry (ITC), and protein crystallography. Inhibitory activity(expressed as ICso) and binding affinity (expressed as dissociationconstant, K_(d)) followed the same trend among the substituents tested(FIG. 14A through FIG. 14I, Table 6). The consistently higher K_(d)values obtained by ITC are likely due to differences in experimentalconditions and detection limits of the activity and binding assays.Compounds 3l, 6i, and 3o were equal to or more potent than thewell-studied Aurora inhibitor VX680, which displayed an ICso value of2.8 nM. For comparison, the in vitro activity of VX680 against Aurora Awas previously determined to IC₅₀=1.4 nM (10) and K_(i)=0.6 nM (8) usingdifferent assays.

TABLE 5 Inhibitory activity and binding affinity of bisanilinopyrimidineinhibitors towards Aurora A.

IC₅₀ ITC^(a) Cmpd PDB R₁ R₂ R₃ (nM)^(a) K_(d) (nM)  3h 3UO5 —H —H —COOH 10 ± 1.6  39 ± 5.9 1 3UP7 —COOH —H —COOH 6.1 ± 1.0  34 ± 5.9  6q 3UO4-phenyl —H —COOH 149 ± 23  299 ± 27   6h 3UOD —CF₃ —H —COOH  35 ± 4.1 49 ± 5.2  6d 3UP2 —OCF₃ —H —COOH  28 ± 4.8  40 ± 5.6  6a 3UNZ —F —H—COOH 3.7 ± 0.7  16 ± 1.6  3l 3UO6 —Cl —H —COOH 2.5 ± 0.3  15 ± 1.5  6i3UOH —Br —H —COOH 2.1 ± 0.4  13 ± 2.2  6n 3U0J —CN —H —COOH  43 ± 8.0 51 ± 5.5  3o 3UOK —Cl —F —COOH 0.8 ± 0.2 n/a 13a 3U0L —Cl —H -Tetrazole3.1 ± 0.4  18 ± 2.7 VX680 2.8 ± 0.3  17 ± 3.7

Experimental data are shown in FIG. 19 through FIG. 20E.

Example 4 Halogen Substituents Induce a DFG-Out State with ConcomitantClosure of the ATP Site

The major difference between the binding interactions of hit compound 1and all other compounds tested is the establishment of a salt bridgebetween the carboxyl group on the A-ring and the side chain of Lys162(FIG. 14A through FIG. 14I and FIG. 21A through FIG. 21D). Lys162normally interacts with the carboxyl group of Asp274, and the loss ofLys162 as a binding partner allows the Asp274 side chain to swingtowards the inhibitor. A similar interaction pattern is found uponbinding of ADP, in which the diphospho moiety interacts with both Lys162and Asp274. The unsubstituted parent compound 3h retains high activityand binding affinity as fewer spatial constraints and loss of theelectrostatic interaction with Lys162 allow the A-ring to rotate byapproximately 40° into a position that is presumably more energeticallyfavorable. Introducing a phenyl group (compound 6q) resulted insignificant reduction of activity due to steric hindrance with the DFGsegment. Although the DFG segment shifts away from the inhibitor toaccommodate the bulky phenyl ring, the DFG-in state is maintained. Inaddition, the A-ring rotates away from the favorable orientation ofcompound 3h. The trifluoromethyl (compound 6h) and trifluoromethoxy(compound 6d) substituents were approximately three-fold less activethan parent compound 3h. These substituents caused steric hindrance withthe DFG as indicated by rotation of the A-ring, particularly forcompound 6d. Binding of compounds 6h and 6d induced a flip of theAla-Asp peptide bond from a cis-like configuration to trans. Thisbackbone structural change is localized and did not alter the DFG-inconformation, but it indicated that the N-terminal flank of the DFG mayrespond to halogen substituents.

Remarkably, introduction of a fluorine (compound 6a), chlorine (compound3l), or bromine (compound 6i) moiety resulted in a significant increaseof inhibitory activity and binding affinity for progressively larger andless electronegative substituents (Table 5). Co-crystallization attemptsusing the established conditions for DFG-in compound 3h, 1, 6q, 6h, and6d were unsuccessful and in-diffusion experiments using crystals ofligand-free Aurora A resulted in rapid deterioration of diffractionpower. These observations suggested that compounds 6a, 3l, and 6i inducestructural changes in the enzyme that are incompatible with the crystallattice. New conditions suitable for the crystallization of Aurora A inthe presence of these inhibitors were established. The resultingstructures revealed a complete switch from the DFG-in to the DFG-outstate (FIG. 14A through FIG. 15E). In the DFG-out state, Asp274 isrotated approximately 100° away from the ATP site and interacts withArg255 and Asn256 (FIG. 15A through FIG. 15E). The main chainconformational change of Asp274 is perpetuated towards Phe275 andGly276, resulting in a complete 180° flip of the DFG motif and theadjacent activation loop (residues 277-293), which moves inward atop theATP cleft. The new loop conformation is stabilized by hydrogen bondinginteractions between the main chain atoms of Lys141 and His280 and bymultiple van-der-Waals interactions of Trp277, which shifts positionfrom a largely polar environment to a strictly hydrophobic pocket (FIG.15D). Another effect of the DFG flip is a ˜4 Å shift in the C-helix toaccommodate Phe275 and movement of the catalytic glutamate residue(Glu181) away from the ATP active site. Notably, VX680 and compound 3hshare the same DFG-in mode of action and exhibit similar conformationsin the DFG segment and the activation loop (FIG. 15E). The closed loopconformation exhibits clearly defined electron density for theN-terminal strand (residues 277-282), which is anti-parallel to aβ-strand of the upper N-terminal lobe of the kinase domain. In contrast,the tip of the loop (residues 283-289) is largely flexible (FIG. 15D).

The high potency of the halogenated compounds 6a, 3l, and 6i appears toresult from structural rearrangement of the activation loop, whicheffectively shields the inhibitor from solvent and ATP in the dead-endcomplex. The increase in enthalpy observed by ITC (Table 5) is likelydue to the addition of hydrogen bonding interactions between the loopand the enzyme upon inhibitor binding. The chlorinated scaffold ofcompound 3l was analyzed by introducing a fluorine in the pyrimidinering (compound 3o), which increased the inhibitory potency by greaterthan two-fold (IC₅₀=0.8 nM). The co-crystal structure confirmed theDFG-out mode of compound 30, and the increased potency over compound 3lis attributable to additional van der Waals interactions of the fluorinesubstituent with the small hydrophobic pocket around the gatekeeperresidue Leu210 (FIG. 16A through FIG. 16C). Substitution of thepara-carboxyl group of the B-ring with a tetrazole moiety (compound 13a)rendered the DFG-out characteristics unchanged, but did not improveinhibitory activity (IC₅₀=3.1 nM). The tetrazole ring mimics the anioniccharacter of the carboxyl group by establishing a salt bridge withArg137. The data demonstrate that substitutions in other regions of thebisanilinopyrimidine scaffold do not affect the DFG-out mode of actionof ortho substituents in the A-ring.

Example 5 The DFG-Out Inhibitors Target Ala273, the Residue N-Terminalto the DFG

The studies of the ortho position of the B-ring show that the observedconformational changes are attributed to the substituents in thisposition. Analysis of the binding interactions of mono-halogenatedcompounds 6a, 3l, and 6i in the respective dead-end complexes did notreveal a reason for the unique conformational changes of the DFG and theactivation loop. The position of the A-ring remains unchanged withrespect to parent compound 3h, and no additional interactions withenzyme residues are observed at first glance. The DFG flip cannot beattributed to steric forces, as the bulky phenyl and trifluoromethoxysubstituents of compounds 6q and 6d did not invoke similar structuralchanges. Furthermore, proximity and net electronegativity alone do notexplain these observations, as binding of the fluorinated substituentsof compounds 6h and 6d renders the DFG-in state unchanged.Superimposition of compound 3l onto the DFG-in state simulates thecollision complex of halogenated inhibitors with the active site priorto the DFG flip (FIG. 17A). Comparison with the dead-end complexindicates that the chlorine atom attracts the methyl group of Ala273,resulting in ˜0.8 Å shorter distance and almost collinear alignment ofthe Phe-Cl and C_(α)-C_(β) bonds. The positional shift of Ala273 towardsthe inhibitor is only observed for the halogenated compounds 6h, 6d, 6a,3l, and 6l and nitrile derivative compound 6n (FIG. 21A through FIG.22D).

Halogen substituents are known for their abilities to significantlyenhance the activity of small molecule inhibitors (Muller et al., (2007)Science 317:1881-1886), but the mechanism for the attraction ofhalocarbons to active site residues is not fully understood. C—X groups(X═F, Cl, Br) frequently display lipophilic characteristics, such asfitting into a hydrophobic pocket as observed for the fluorinesubstituent of compound 3o (FIG. 16A through FIG. 16B). Recent analysesof the PDB revealed a large number of halogenated ligands that appear toestablish polar “halogen bonding” interactions with their targetproteins (Lu et al., (2009) J Med Chem 52:2854-2862; Lu et al., (2009) JPhys Chem. B 113:12615-12621; Parisini et al., (2011) Halogen bonding inhalocarbon-protein complexes: a structural survey, Chem Soc Rev40:2267-2278). Non-covalent halogen bonds are weaker than hydrogenbonding interactions, and they are typically established with polaracceptor groups in the form of perpendicular C—X . . . H or linear C—X .. . D bonds, in which D (electron donor) is a Lewis base. Halogenbonding is a potential mechanism for the DFG-out inhibitors, taking intoaccount the concept of the ‘sigma-hole’ (Auffinger et al., (2004) ProcNatl Acad Sci US A 101:16789-16794), which describes the positiveelectrostatic potential at the tip of the C—X bond due to the unevenlydistributed partial charges around the halogen atom (Cl, Br, and I). C—Xbonds therefore assume either electrophilic or nucleophiliccharacteristics, depending on the geometry of the halogen bond (Politzeret al., (2007) J Mol Model 13:305-311; Murray et al., (2009) J Mol Model15:723-729). Halogen bonding is highly directional, with the C—X . . . Dbond typically existing in a linear alignment (angles between 140-1800)(Metrangolo et al., (2001) Chemistry 7:2511-2519; Metrangolo et al.,(2008) Angew Chem Int Ed Engl 47:6114-6127; Politzer et al., (2010) PhysChem Chem Physics: PCCP 12:7748-7757). This implies that the strength ofthe sigma-hole halogen bond is directly related to the C—X . . . Dalignment, with the strongest orbital overlap in the linear orientationand deviation from 180° resulting in a partial to total loss of effect.In the case of the DFG-out inhibitors, however, several experimentalobservations render halogen bonding interactions an unlikely cause forthe DFG flip. First, the sigma-hole concept may be applicable to thechlorine and bromine substituents of compounds 3l and 6i, but not to thefluorine of compound 6a (Parisini et al., (2011) Chem Soc Rev40:2267-2278). Second, the methyl group of Ala273 is a poor Lewisbase/electron donor for an interaction of this nature. Third, halogenbonding interactions contribute to the binding potential of smallmolecules only moderately, and they have not been associated withconformational changes in proteins. The magnitude of the structuralchanges in Aurora A induced by compounds 6a, 3l, and 6i thereforesuggested a different mechanism of action involving electrostaticdipole-dipole interactions.

Example 6 Induced-Dipole Forces Likely Cause the DFG to Unwind

The fundamental concept that polar molecules have the ability topolarize a second, nonpolar molecule has been given much attention inmolecular dynamics simulations to understand and predict the influenceof electrostatic and van-der-Waals forces in proteins (Neves-Petersen etal., (2003) Biotechnol Ann Rev 9:315-395; Stork et al., (2007) J ChemPhys 126:165106; Nakamura (1996) Q Rev Biophys 29:1-90). The asymmetricpacking of the main chain amide dipoles in folded proteins results inoverall positive electrostatic potential of all side chains,particularly for alanine (Gunner et al., (2000) Biophys J 78:1126-1144).The dipole moment and molecular orbital signature of L-alanine has beenthoroughly analyzed (Falzon et al., (2006) J Phys Chem B 110:9713-9719),and the polarizability of Ala273 in Aurora A may therefore play animportant role in the mode of action of the inhibitors. The Phe-X bondsof compound 6a, 3l, and 61 align with the C_(α)-C_(β) bond of Ala273,reminiscent of the attraction exerted by a permanent magnet on an ironrod (FIG. 17B). In this mechanism, the electric dipole of the inhibitorinduces a dipole in the polarizable C_(α)-C_(β) bond of Ala273.Establishment of this dipole-dipole interaction is relayed to theπ-system of the amide bond, altering the charge distribution along theDFG backbone and allowing unrestrained rotation. As a consequence, theDFG unwinds and the activation loop adopts a potentially lower energystate. Such an unprecedented mechanism of action is difficult to prove,and computational drug design has not probed the effect of exogenouselectric dipoles on protein structure (Lu et al., (2009) J Phys Chem B113:12615-12621).

To test the hypothesis of induced-dipole forces being responsible forthe DFG flip, and to rule out halogen bonding as a plausible mechanism,the strong dipolar and electron-withdrawing nitrile group was introduced(Jones et al., (2010) Med Chem Comm 1:309-318; Fleming et al., (2010) JMed Chem 53:7902-7917). Compound 6n induced the same structural changesas compounds 6a, 3l, and 6i (FIG. 14A through FIG. 14I), demonstratingthat the DFG-in state of Aurora A is readily perturbed by dipoles ableto align with Ala273. The distances between the substituents and the Cβatom of Ala273 range from 3.9 Å for the fluorine to as low as 3.0 Å forthe nitrile substituent (FIG. 17C). While the respective halocarbonbonds are positioned almost collinearly with the C_(α)-C_(β) bond ofAla273, accommodation of the longer nitrile group necessitates rotationof the A-ring out of the energetically favored position by ˜20°.Although slightly misaligned, the close proximity of the nitrile dipoleto Ala273 induces the same conformational changes in the enzyme as thehalogen substituents. However, steric repulsion exerted by Ala273 causesstrain in the inhibitor molecule explaining the decrease of bindingpotential for compound 6n (Table 5). The inhibitory efficacy of DFG-outinhibitors harboring electric dipoles depends on a precise geometrybetween the substituents and the flanking alanine residue. AlthoughAla273 is in close distance to compounds 4 and 5, the dipoles introducedby the —CF₃ and —OCF₃ substituents lack the potential to induce the DFGflip. The C—F bonds are positioned almost orthogonal to the C_(α)-C_(β)bond, and steric repulsion prevents the proper alignment of the overalldipole along the Phe-CF₃ axis of compound 4 with Ala273 (˜150° angle and4 Å distance).

Example 7 Implications for the Design of DFG-Out Inhibitors of OtherKinases

The DFG-out inhibitors designed herein are among the most potentdescribed for Aurora A and protein kinases in general. The ADFG sequenceand the three-dimensional architecture of the DFG-in state arewell-conserved among kinases for which structural information isavailable (Table 6, FIG. 18A and FIG. 18B). It is conceivable thatkinases with highest structural similarity to the DFG-in state of AuroraA respond similarly to the introduction of electric dipoles directed atthe flanking alanine residue. The bisanilinopyrimidine scaffold ofcompound 1 proved to be advantageous for the design of DFG-outinhibitors of Aurora A, as it already binds with high affinity to theDFG-in state. However, slight variations in the ATP site may preventproper positioning of this scaffold to other kinases. For example,compounds 1 and 7 are poor inhibitors of CDK2 with IC₅₀ values of 11 and15 μM, respectively. With the exception of CDK8 (Schneider et al.,(2011) J Mol Biol 412:251-266), CDKs are known only in the DFG-in state,despite the numerous inhibitor scaffolds that have been discovered. Theconformation of the C-terminal DFG flank in CDK2 differs significantlyfrom most kinases and can contribute to the stabilization of the DFG-instate. Modeling of the conformer of compound 7 (Aurora A) into theactive site of CDK2 indicates substantial steric hindrance caused by theA-ring (FIG. 18C). Co-crystal structures revealed that these inhibitorsbind to CDK2 in the (s)-trans conformation as opposed to the (s)-cisconformation adopted in Aurora A (FIG. 18D). As a result, the A-ringprojects away from the DFG, preventing dipolar substituents from linearalignment with Ala273. Therefore, targeting the DFG of other kinases byinduced-dipole forces initially requires the identification of candidatescaffolds that satisfy the geometric framework for efficient interactionwith the N-terminal flank.

TABLE 6 Structural comparison of the DFG-in states of selected kinasesharboring an ADFG sequence. Overall identity/ PDB ID similarity (%)^(a)Kinase r.m.s.d. (Å)^(b) 2HK5 22.3/41.1 HCK 0.2 2DQ7 23.8/40.9 Fyn 0.21QPC 23.7/39.0 LCK 0.2 2ZV7 23.8/40.2 Lyn 0.2 3KF4 24.8/42.6 ABL1 0.32EU9 19.9/36.5 CLK3 0.3 3COK 35.1/56.2 PLK4 0.3 2XIK 30.3/49.0 STK25 0.32J7T 28.3/46.9 STK10 0.3 3A7F 28.6/48.1 STK24 0.3 3COM 26.8/49.7 STK40.3 2QLU 21.7/41.5 ACTR-IIB 0.3 2JS1 28.5/49.3 hSLK 0.3 3S95 22.8/39.9LIMK1 0.3 3LXL 24.6/42.4 JAK3 0.3 2BDJ 22.9/40.5 Src 0.3 1XJD 30.8/49.3PKC theta 0.4 2VD5 30.8/48.4 DMPK 0.4 2ETR 30.2/48.2 ROCK 1 0.5 2VZ631.3/48.1 CaMK2A 0.5 3DAK 25.9/41.3 OSR1 1.0 3PXR 26.4/44.4 CDK2 1.03FE3 31.2/51.5 MAPK3 1.0 3LMG 20.8/39.1 erbB-3 1.0 3KY2 25.4/42.2 FGFR11.1 3L8P 25.2/40.2 Tie-2 1.2 3PLS 21.3/35.4 RON 1.2 3BRB 22.9/36.7 MER1.3 1BLX 24.3/41.0 CDK6 1.3 3F66 21.1/36.0 c-Met 1.4 ^(a)Computed withEMBOSS Needle against the kinase domain of human Aurora A. ^(b)r.m.s.d.= root mean square deviation of the main chain atoms upon superpositionwith the ADFG of Aurora A in complex with compound 1 (computed withSuperpose from the CCP4 program suite).

Example 8 Compounds

All reagents were purchased from commercial suppliers and used withoutfurther purification. Melting points were determined using a Bamsteadinternational melting point apparatus and remain uncorrected. ¹H NMRspectra were recorded on a Varian Mercury 400 MHz spectrometer withCDCl₃ or DMSO-d₆ as the solvent. ¹³C NMR spectra are recorded at 100MHz. All coupling constants are measured in Hertz (Hz) and the chemicalshifts (δ_(H) and δ_(C)) are quoted in parts per million (ppm) relativeto TMS (δ 0), which was used as the internal standard. High resolutionmass spectroscopy was carried out on an Agilent 6210 LC/MS (ESI-TOF).Microwave reactions were performed in CEM 908005 model and Biotageinitiator 8 machines. HPLC analysis was performed using a JASCO HPLCsystem equipped with a PU-2089 Plus quaternary gradient pump and aUV-2075 Plus UV-VIS detector, using an Alltech Kromasil C-18 column(150×4.6 mm, 5 μm) and Agilent Eclipse XDB-C18 (150×4.6 mm, 5 μm).Melting points were recorded on an Optimelt automated melting pointsystem (Stanford Research Systems). Thin layer chromatography wasperformed using silica gel 60 F254 plates (Fisher), with observationunder UV when necessary. Anhydrous solvents (acetonitrile,dimethylformamide, ethanol, isopropanol, methanol and tetrahydrofuran)were used as purchased from Aldrich. Burdick and Jackson HPLC gradesolvents (methanol, acetonitrile and water) were purchased from VWR forHPLC and mass analysis. HPLC grade TFA was purchased from Fisher.

Synthetic Protocols for 2a to 2o:

N⁴-(2-Carboxyphenyl)-2-chloropyrimidine-4-amine hydrochloride (2a,method a in FIG. 4A)

A mixture of 2,4-dichloropyrimidine (0.149 g, 1.00 mmol) and2-aminobenzoic acid (0.137 g, 1.00 mmol) in HCl (3.0 mL, 0.1 M) washeated in a microwave reactor at 100° C. for 30 min. The precipitateobtained was filtered and the product washed with water (10 mL), hotMeOH (2×10 mL) and dried under vacuum to provide 2a (0.195 g, 68%) as alight yellow solid. m.p. 169° C. (dec.). ¹H NMR (400 MHz, DMSO-d₆) δ10.70 (s, 1H), 8.21 (d, J=6.0 Hz, 1H), 8.13 (d, J=8.0 Hz, 1H), 7.95 (dd,J=8.0, 1.6 Hz, 1H), 7.65-7.60 (m, 1H), 7.20 (appt, J=7.6 Hz, 1H), 6.90(d, J=5.6 Hz, 1H); LRMS (ESI−) m/z 248.0 (M−H−HCl)⁻; HRMS (ESI+) m/zcalculated for C₁₁H₉CN₃O₂ (M−Cl)⁺ 250.0378. found 250.0380.

N⁴-(2-Carbamoylphenyl)-2-chloropyrimidine-4-amine (2b, method a in FIG.4A)

A mixture of 2,4-dichloropyrimidine (0.149 g, 1.00 mmol) and2-aminobenzamide (0.136 g, 1.00 mmol) in HCl (3.0 mL, 0.1 M) was heatedin a microwave reactor at 100° C. for 30 min. The precipitate obtainedwas filtered and the product was washed with water (10 mL) andrecrystallized in methanol to obtain pure 2b (0.179 g, 63%) as a lightyellow solid. m.p. 197° C. (dec.). ¹H NMR (400 MHz, DMSO-d₆) δ 11.24 (s,1H), 8.21 (brs, 1H partially overlapping), 8.19 (d, J=5.6 Hz, 1Hpartially overlapping), 8.15 (d, J=8.0 Hz, 1H partially overlapping),7.75 (dd, J=8.4, 1.6 Hz, 1H), 7.71 (brs, 1H), 7.55-7.51 (m, 1H), 7.16(td, J=7.6, 1.2 Hz, 1H), 6.85 (d, J=5.6 Hz, 1H); LRMS (ESI+) m/z 249.0(M-Cl)⁺; HRMS (ESI+) m/z calculated for C₁₁H₁₀ClN₄O (M−Cl)⁺ 249.0538.found 249.0545.

N⁴-Phenyl-2-chloropyrimidine-4-amine hydrochloride (2c, method a in FIG.4A)

A mixture of 2,4-dichloropyrimidine (0.317 g, 2.12 mmol) and aniline(0.198 g, 2.12 mmol) in HCl (6.0 mL, 0.1 M) was heated in a microwavereactor at 100° C. for 30 min. The product obtained was purified usingSiO₂ chromatography to afford the desired compound 2c (0.130 g, 26%) asa white solid. m.p. 159-161° C. ¹H NMR (400 MHz, CD₃OD) δ 8.03 (dd,J=6.0, 1.0 Hz, 1H), 7.57 (d, J=8.0 Hz, 2H), 7.37-7.33 (m, 2H), 7.12 (t,J=7.6 Hz, 1H), 6.67 (dd, J=6.0, 1.0 Hz, 1H); LRMS (ESI+) m/z 206.0(M-Cl)⁺; HRMS (ESI) m/z calculated for C₁₀H₉ClN₃ (M−Cl)⁺ 206.0480. found206.0477.

N⁴-(4-Carboxyphenyl)-2-chloropyrimidine-4-amine hydrochloride (2d,method a in FIG. 4A)

A mixture of 2,4-dichloropyrimidine (0.596 g, 4.00 mmol) and4-aminobenzoic acid (0.549 g, 4.00 mmol) in HCl (12.0 mL, 0.1 M) washeated in a microwave reactor at 100° C. for 30 min. The precipitateobtained was filtered and the product was washed with water (10 mL×2)and dried under high vacuum to give the desired compound 2d (0.895 g,78%) as a white solid. m.p. 244° C. (dec.). ¹H NMR (400 MHz, DMSO-d₆) δ10.38 (s, 1H), 8.24 (d, J=6.0 Hz, 1H), 7.92 (d, J=8.8 Hz, 2H), 7.73 (d,J=8.8 Hz, 2H), 6.85 (d, J=5.6 Hz, 1H); LC-MS (ESI+) m/z 250.03 (M-Cl)⁺;HRMS (ESI+) m/z calculated for C₁₁H₉ClN₃O₂ (M-Cl)⁺ 250.0378. found250.0378.

N⁴⁻⁽2-Carbomethoxyphenyl)-2-chloropyrimidine-4-amine hydrochloride (2e,method a in FIG. 4A)

A mixture of 2,4-dichloropyrimidine (0.149 g, 1.00 mmol) and methyl2-aminobenzoate (0.151 g, 1.00 mmol) in HCl (3.0 mL, 0.1 M) was heatedusing a microwave reactor at 100° C. for 30 min. The pale yellow solidwhich precipitated upon cooling was filtered and washed with water (10mL). The solid was then dissolved in hot EtOH (20 mL) and filtered toremove the insoluble impurity. The filtrate was concentrated to obtainthe desired product 2e (0.135 g, 45%) as a pale yellow solid. m.p.: 119°C. (dec). ¹H NMR (400 MHz, DMSO-d₆) 10.24 (s, 1H), 8.18 (d, J=6.0 Hz,1H), 7.85 (dd, J=7.6, 1.6 Hz, 1H), 7.79 (d, J=8.4 Hz, 1H), 7.64-7.60 (m,1H), 7.25 (appt, J=7.6 Hz, 1H), 6.82 (d, J=6.0 Hz, 1H), 3.74 (s, 3H);LRMS (ESI+) m/z 264.0 (M−Cl)⁺; HRMS (ESI+) m/z calculated forC₁₂H₁₂ClN₃O₂ (M−Cl)⁺ 264.0534. found 264.0542.

N⁴-(2-Carboxyphenyl)-2-chloro-6-methylpyrmidine-4-amine hydrochloride(2f, method a in FIG. 4A)

A mixture of 2,4-dichloro-6-methylpyrimidine (1.630 g, 0.010 mol) and2-aminobenzoic acid (1.370 g, 0.010 mol) in HCl (15.0 mL, 0.1 M) washeated in a microwave reactor at 100° C. for 30 min. The precipitateobtained was filtered and washed with water (10 mL×2), MeOH (10 mL×2)and acetone (5 mL×2) sequentially. The product obtained was dissolved inhot EtOH (30 mL), filtered and the filtrate was concentrated andrecrystallized in MeOH to afford the desired compound 2f (1.240 g, 41%)as a white solid. m.p. 169° C. (dec.). ¹H NMR (400 MHz, DMSO-d₆) δ 10.71(s, 1H), 8.21 (d, J=8.4 Hz, 1H), 7.96 (dd, J=7.9, 1.5 Hz, 1H), 7.63(appt, J=7.2 Hz, 1H), 7.16 (appt, J=7.6 Hz, 1H), 6.80 (s, 1H), 2.31 (s,3H); LC-MS (ESI−) m/z 262.03 (M−H−HCl)⁻; HRMS (ESI−) m/z calculated forC₁₂H₉ClN₃O₂ (M−H−HCl)⁻ 262.0389. found 262.0393.

N⁴-(2-Carboxyphenyl)-2-chloro-5-fluoropyrimidine-4-amine hydrochloride(2g, method a in FIG. 4A)

A suspension of 2,4-dichloro-5-fluoropyrimidine (0.835 g, 5.00 mmol) and2-aminobenzoic acid (0.685 g, 5.00 mmol) in HCl (15.0 mL, 0.1 M) washeated in a microwave reactor at 100° C. for 30 min. The precipitateobtained was filtered and washed with water (10 mL×2). The productobtained was slurried in hot MeOH (10 mL×2), filtered and dried underhigh vacuum to afford the desired compound 2g (0.836 g, 55%) as a lightyellow solid. m.p. 194-197° C. ¹H NMR (400 MHz, DMSO-d₆) 11.67 (s, 1H),8.57 (d, J=8.4 Hz, 1H), 8.44 (d, J=3.0 Hz, 1H), 8.03 (dd, J=8.0, 1.6 Hz,1H), 7.71 (appt, J=7.8 Hz 1H), 7.21 (t, J=7.6 Hz, 1H); ¹⁹F NMR (376 MHz,DMSO-d₆) δ −155.07 (appt d, J=2.4 Hz); LC-MS (ESI−) m/z 266.01(M−H−HCl)⁻; HRMS (ESI−) m/z calculated for C₁₁H₆ClFN₃O₂ (M−H−HCl)⁻266.0138. found 266.0143.

N⁴-(2-Carboxyphenyl)-2-chloropyrimidine-4,5-diamine hydrochloride (2h,method a in FIG. 4A)

A mixture of 2,4-dichloropyrimidin-5-amine (0.270 g, 1.646 mmol) and2-aminobenzoic acid (0.225 g, 1.646 mmol) in HCl (4.0 mL 0.1 M) washeated in a microwave reactor at 100° C. for 30 min. The precipitateobtained was filtered and washed with water (5 mL), then with MeOH (3mL, quick wash) and dried to afford the desired compound 2h (0.280 g,57%) as a yellow solid. m.p. 172° C. (dec.). ¹H NMR (400 MHz, DMSO-d₆) δ13.45 (brs, 1H), 10.76 (s, 1H), 8.49 (d, J=8.5 Hz, 1H), 8.00 (dd, J=7.9,1.4 Hz, 1H), 7.80 (s, 1H), 7.63 (appt, J=7.2 Hz, 1H), 7.11 (appt d,J=7.6 Hz, 1H), 5.04 (brs, 2H); LC-MS (ESI+) m/z 265.06 (M-Cl)⁺; HRMS(ESI+) m/z calculated for C₁₁H₁₀ClN₄O₂ (M−Cl)⁺ 265.0487. found 265.0492.

N⁴-(2-Carboxyphenyl)-2-chloropyrimidine-4,6-diamine hydrochloride (2i,method c in FIG. 4A)

A mixture of 2,6-dichloropyrimidin-4-amine (2.006 g, 12.23 mmol) and2-aminobenzoic acid (1.676 g, 12.23 mmol) in HCl (18.0 mL, 0.1 M) washeated at 100° C. in a sealed pressure tube for 24 h. The suspension wasfiltered after cooling to r.t. and washed with water (5 mL) and acetone(5 mL). The product obtained was slurried with acetone (10 mL), MeOH (10mL), and again with acetone (10 mL) sequentially to afford pure compound2i (1.562 g, 43%) as a light brown solid. m.p. 207-210° C. ¹H NMR (400MHz, DMSO-d₆) δ 10.89 (s, 1H), 8.83 (d, J=8.4 Hz, 1H), 7.97 (d, J=7.6Hz, 1H), 7.54 (t, J=6.8 Hz, 1H), 7.18 (s, 2H), 6.99 (t, J=7.2 Hz, 1H),6.02 (s, 1H); LC-MS (ESI+) m/z 265.06 (M-Cl)⁺; HRMS (ESI+) m/zcalculated for C₁₁H₁₀ClN₄O₂ (M−Cl)⁺ 265.0487. found 265.0488.

N⁴-(2-Carboxyphenyl)-2-chloro-5-methylpyrimidine-4-amine hydrochloride(2j, method a in FIG. 4A)

A mixture of 2,4-dichloro-5-methylpyrimidine (0.535 g, 3.28 mmol) and2-aminobenzoic acid (0.450 g, 3.28 mmol) in HCl (6.0 mL of 0.1 M) washeated in a microwave reactor at 100° C. for 30 min. The productobtained was filtered and washed with water (10 mL), slurried using hotMeOH (10 mL). The product was filtered and dried to obtain the desiredcompound 2j (0.512 g, 52%) as a light yellow solid. m.p. 184° C. (dec.).¹H NMR (400 MHz, DMSO-d₆) δ 11.28 (s, 1H), 8.76 (d, J=8.5 Hz, 1H), 8.20(s, 1H), 8.04 (dd, J=8.0, 1.5 Hz, 1H), 7.68 (t, J=7.2 Hz, 1H), 7.16 (t,J=7.6 Hz, 1H), 2.20 (s, 3H); LC-MS (ESI+) m/z 264.05 (M-Cl)⁺; HRMS(ESI+) m/z calculated for C₁₂H₁₁ClN₃O₂ (M-Cl)⁺ 264.0534. found 264.0539.

N⁴-(2-Chlorophenyl)-2-chloro-5-fluoropyrimidine-4-amine hydrochloride(2k, method b in FIG. 4A)

A solution of 2,4-dichloro-5-fluoropyrimidine (2.00 g, 1.19 mmol) and2-chloroaniline (1.53 g, 1.99 mmol) in HCl (0.1 M, 40.0 ml) were stirredat rt for 5 days. The precipitate obtained was filtered, washed withwater and dried to obtain 2k.HCl (1.60 g, 47%) as a white solid. m.p.128-129° C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.98 (s, 1H), 8.30 (d, J=3.4 Hz,1H), 7.57 (dd, J=7.9, 1.5 Hz, 1H), 7.47 (dd, J=7.8, 1.7 Hz, 1H), 7.40(td, J=7.6, 1.6 Hz, 1H), 7.34 (td, J=7.6, 1.8 Hz, 1H); ¹³C NMR (101 MHz,DMSO) δ 156.9 (d, J=3.1 Hz), 152.97 (d, J=12.3 Hz), 145.82 (d, J=257.5Hz), 142.55 (d, J=20.5 Hz), 134.70, 131.40, 130.55, 129.86, 129.10,128.49; ¹⁹F NMR (376 MHz, DMSO) δ −155.74 (s); LC-MS (ESI+) m/z 258.0001(M−Cl)⁺; HRMS (ESI+) m/z calculated for C₁₀H₇Cl₂FN₃ (M-Cl)⁺ 257.9996.found 258.0011.

N⁴-(2-Chlorophenyl)-2-chloropyrimidine-4-amine (21, method b in FIG. 4A)

A solution of 2,4-dichloropyrimidine (1.90 g, 1.3 mmol) and2-chloroaniline (1.63 g, 1.3 mmol) in HCl (0.1 M, 40.0 mL) were stirredat RT for 3 days. The precipitate obtained was filtered and washed withsat. sodium bicarbonate solution and water. The product was purified bya quick wash with ether, dried to obtain 2l (2.70 g, 87%) as a whitesolid. Mp: 116-117° C.; ¹H NMR (400 MHz, DMSO-d₆) δ 9.75 (s, 1H), 8.13(d, J=5.9 Hz, 1H), 7.59 (dd, J=8.0, 1.4 Hz, 1H), 7.55 (dd, J=8.0, 1.4Hz, 1H), 7.38 (td, J=7.8, 1.6 Hz, 1H), 7.27 (td, J=7.8, 1.6 Hz, 1H),6.65 (d, J=5.8 Hz, 1H). ¹³C NMR (100 MHz, DMSO) δ 163.19, 160.18,158.34, 135.32, 130.64, 129.42, 128.53, 128.25, 128.11, 105.60. LC-MS(ESI+) m/z 240.01 (M−H)⁻; HRMS (ESI+) m/z calculated for C₁₀H₇Cl₂N₃(M+H)⁺ 240.0090. found 240.0090.

N⁴-(2-Carboxyphenyl)-2,5-dichloropyrimidine-4-amine hydrochloride (2m,method b in FIG. 4A)

A mixture of 2,4,5-trichloropyrimidine (0.156 g, 0.852 mmol) and2-aminobenzoic acid (0.116 g, 0.852 mmol) in HCl (0.1 M aq., 3.0 mL),was heated in a microwave reactor at 100° C. for 30 min. The resultingprecipitate was filtered and washed with water (3 mL×2), Et₂O (3 mL×2),DCM (5 mL×2) and EtOAc (5 mL) sequentially and dried under vacuum toafford the title compound 2m (0.114 g, 42%) as a white solid. m.p.209.1-210.4° C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.80 (s, 1H), 8.64 (d,J=8.4 Hz, 1H), 8.53 (s, 1H), 8.05 (dd, J=1.1, 7.9 Hz, 1H), 7.72-7.68 (m,1H), 7.23 (t, J=7.8 Hz, 1H); LC-MS (ESI−) m/z 281.99 (M−H−HCl)⁻; HRMS(ESI−) m/z calculated for C₁₁H₆Cl₂N₃O₂ (M−H−HCl)⁻ 281.9843. found281.9839.

N⁴-(2-Carboxyphenyl)-2,5,6-trichloropyrimidine-4-amine hydrochloride(2n, method b in FIG. 4A)

A mixture of 2,4,5,6-tetrachloropyrimidine (0.963 g, 4.45 mmol) and2-aminobenzoic acid (0.610 g, 1.48 mmol) in HCl (0.1 M aq., 14.0 mL) washeated in a microwave reactor at 100° C. for 30 min. The resultingprecipitate was filtered and washed with water (5 mL×2), dried andslurried in methanol (5 mL). The solid was filtered and dried undervacuum to afford the title compound 2n (0.513 g, 32%) as a white solid.m.p. 230° C. (dec.). ¹H NMR (400 MHz, DMSO-d₆) δ 11.88 (s, 1H), 8.53(dd, J=0.8, 8.4 Hz, 1H), 8.05 (dd, J=1.6, 7.9 Hz, 1H), 7.73-7.79 (m,1H), 7.70-7.23 (m, 1H); LC-MS (ESI−) m/z 315.94 (M−H−HCl)⁻; HRMS (ESI−)m/z calculated for C₁₁H₅Cl₃N₃O₂ (M−H−HCl)⁻ 315.9453. found 315.9454.

N⁴-(2-Chlorophenyl)-2,5-dichloropyrimidine-4-amine (20, method b in FIG.4A)

A solution of 2,4-dichloro-5-fluoropyrimidine (2.00 g, 10.9 mmol) and2-chloroaniline (1.53 g, 12.0 mmol) in HCl (0.1 M, 40 ml) were stirredat RT for 5 days. The precipitate obtained was filtered, washed withwater and sodium bicarbonate (2 M) and dried to obtain 2o (1.60 g, 47%)as a white solid. m.p. 128-129° C. ¹H NMR (400 MHz, CDCl₃) δ 8.58 (s,1H), 8.50 (dd, J=8.4, 1.6 Hz, 1H), 7.98 (brs, 1H), 7.43 (dd, J=8.0, 1.2Hz, 1H), 7.36 (apparent td J=7.6, 1.6 Hz, 1H), 7.10 (td, J=7.6, 1.2 Hz,1H); LC-MS (ESI−) m/z 273.97 (M+H)⁺; HRMS (ESI+) m/z calculated forC₁₀H₆Cl₃N₃ (M+H)⁺ 273.9700. found 273.9700 9708.

2-Chloro-N-(2,6-dichlorophenyl)-5-fluoropyrimidin-4-amine (2p, method kin FIG. 5A)

To a solution of 2,6-dichloroaniline (0.58 g, 3.89 mmol) in DMF (10 ml)under argon was added NaH (0.47 g, 19.45 mmol) and stirred for 30 min.To this mixture was added 2,4-dichloro-5-fluoropyrimidine (0.65g, 3.89mmol) and stirred for 14 h. The reaction was queched with water andextracted with DCM (2×15 ml). DCM extracts were washed with water (5×15ml). Organic solvent was dried (MgSO₄), evaporated and the residue waspurified by column chromatography (gradient elution with EtOAc: hexane)to provide 2p as a yellow solid. (421 mg, 37%); ¹H NMR (400 MHz, CDCl₃)δ 8.11 (d, J=2.6 Hz, 1H), 7.42 (d, J=8.1 Hz, 2H), 7.29-7.16 (m, 1H),6.66 (s, 1H); LC-MS (ESI+) m/z 291.95 (M+H)⁺; HRMS (ESI+ve) m/zcalculated for C₁₀H₆Cl₃FN₃ (M+H)⁺ 291.9606. found 291.9607.

2-Chloro-5-fluoro-N-(2-nitrophenyl)pyrimidin-4-amine (2q, method k inFIG. 5A)

This compound was synthesized using the same protocol for 2p exceptusing 2-nitroaniline (0.35 g, 2.53 mmol),2,4-dichloro-5-fluoropyrimidine (0.42 g, 2.53 mmol) and NaH (0.30g,12.65 mmol). The compound 2q was obtained as a bright yellow solid. (217mg, 32%). ¹H NMR (400 MHz, CDCl₃) δ 10.79 (s, 1H), 8.98 (dd, J=8.6, 1.2Hz, 1H), 8.31 (dd, J=8.5, 1.6 Hz, 1H), 8.24 (d, J=2.3 Hz, 1H), 7.76(ddd, J=8.7, 7.4, 1.5 Hz, 1H), 7.29-7.18 (m, 1H); LC-MS (ESI+) m/z269.02 (M+H)⁺; HRMS (ESI+ve) m/z calculated for C₁₀H₇CFN₄O₂ (M+H)⁺269.0236. found 269.0241.

Synthetic Protocols for 5a to 5k:

N⁴-(2-Fluorophenyl)-2-chloropyrimidine-4-amine (5a, method i in FIG. 5A)

A mixture of 2,4-dichloropyrimidine (0.24 g, 1.61 mmol), 2-fluoroaniline(0.16 mL, 1.67 mmol) and DIPEA (0.308 mL, 1.77 mol) in n-butanol (1.0mL) was stirred at 125° C. in sealed tube for 20 h. The solution wasdiluted with water (4 mL) and extracted with ethyl acetate (5 mL×2). Theorganic phase was dried over Na₂SO₄, filtered and washed with ethylacetate. The filtrate was concentrated and the residue was purified withflash chromatography (10 g silica gel, hex/EtOAc) to afford 5a (0.213 g,59%) as a white solid. m.p. 117.9-119.0° C. ¹H NMR (400 MHz, DMSO-d₆) δ9.83 (s, 1H), 8.17 (d, J=5.9 Hz, 1H), 7.74 (apparent t, 1H), 7.35-7.27(m, 1H), 7.26-7.19 (m, 2H), 6.76 (d, J=5.6 Hz, 1H); LC-MS (ESI−) m/z224.04 (M+H)⁺; HRMS (ESI+) m/z calculated for C₁₀H₅ClFN₃ (M+H)⁺224.0385. found 224.0385.

N⁴-(2-Chloro-4-fluorophenyl)-2-chloropyrimidine-4-amine (5b, method i inFIG. 5A)

This was prepared using 2,4-dichloropyrimidine (0.239 g, 1.60 mmol),2-chloro-4-fluoroaniline (0.2 mL, 1.67 mmol) and DIPEA (0.305 mL, 1.75mmol) to afford 5b (0.192 g, 46%) as an off-white solid which was usedwithout further purification. ¹H NMR (400 MHz, DMSO-d₆) δ 9.75 (s, 1H),8.14 (d, J=5.9 Hz, 1H), 7.68-7.47 (m, 2H), 7.33-7.25 (m, 1H), 6.62 (d,J=5.9 Hz, 1H); LC-MS (ESI+) m/z 258.01 (M+H)⁺; HRMS (ESI+) m/zcalculated for C₁₀H₇Cl₂FN₃ (M+H)⁺ 258.9996. found 258.9990.

N⁴-(2-(Trifluoromethoxy)phenyl)-2-chloropyrimidine-4-amine (5c, method iin FIG. 5A)

This was prepared using 2,4-dichloropyrimidine (0.246 g, 1.651 mmol),2-(trifluoromethoxy)aniline (0.3 mL, 2.20 mmol) and DIPEA (0.315 mL,1.81 mmol) to afford 5c (0.161 g, 50%) as an off-white solid. m.p.92.0-94.8° C. ¹H NMR (400 MHz, CDCl₃) δ 8.20 (d, J=5.9 Hz, 1H), 7.89 (d,J=7.9 Hz, 1H), 7.42-7.31 (m, 2H), 7.26-7.20 (m, 1H), 6.62 (d, J=5.9 Hz,1H); LC-MS (ESI+) m/z 290.04 (M+H)⁺; HRMS (ESI+) m/z calculated forC₁₁H₈F₃ClNN₃O (M+H)⁺ 290.0303. found 290.0300.

N⁴-(2-Methoxyphenyl)-2-chloropyrimidine-4-amine (5d, method j, FIG. 5A)

A mixture of 2,4-dichloropyrimidine (2.569 g, 17.24 mmol),2-methoxyaniline (1.9 mL, 16.19 mmol) and Na₂CO₃ (5.781 g, 54.54 mmol)in n-BuOH (36.0 ml) was heated at 100° C. under Argon for 24 h. Themixture was filtered and the resulting precipitate was washed with ethylacetate (10 mL). The combined filtrates were concentrated to dryness toproduce a brown solid. The solid was slurried with ethyl acetate (20mL), filtered and washed with ethyl acetate (5 mL×2), then hexane (20mL) to afford 5d (2.458 g, 60%) as a light brown solid. m.p.126.3-129.2° C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.37 (s, 1H), 8.06 (d, J=5.9Hz, 1H), 7.64 (d, J=7.3 Hz, 1H), 7.21-7.13 (m, 1H), 7.08 (dd, J=8.3, 1.3Hz, 1H), 6.95 (td, J=7.7, 1.4 Hz, 1H), 6.66 (apparent s, 1H), 3.80 (s,3H); LC-MS (ESI+) m/z 236.06 (M+H)⁺; HRMS (ESI+) m/z calculated forC₁₁H₁₁ClN₃O M+H)⁺ 236.0585. found 236.0588.

N⁴-(2-Cyanophenyl)-2-chloropyrimidine-4-amine (5e, method k in FIG. 5A)

To a solution of 2-cyanoaniline (0.543 g, 4.59 mmol) in anhydrous DMF(6.0 mL), NaH (60% dispersion in mineral oil, 0.384 g, 10.77 mmol) wasadded portion wise at 0° C., followed by the addition of2,4-dichloropyrimidine (0.612 g, 4.10 mmol) under positive Argonpressure. The reaction mixture was slowly warmed to room temperature andstirred for a further for 24 h. Water (50 ml) was added and theresulting precipitate was filtered and washed with water (10 mL×2). Thedried solid was dissolved in acetone (40 ml), activated charcoal (ca. 1g) was added and the mixture was stirred for 10 min at room temperature.The activated charcoal was removed by filtration through a celite/silicabed. The filtrate was collected and the solvent removed under reducedpressure to provide the title compound 5e (0.218 g, 22%) as an off-whitecolor solid. m.p. 160° C. (dec). ¹H NMR (400 MHz, DMSO-d₆) δ 10.23 (s,1H), 8.21 (d, J=5.8 Hz, 1H), 7.86 (dd, J=1.3, 7.8 Hz, 1H), 7.74-7.70 (m,1H), 7.62 (d, J=7.8 Hz, 1H), 7.38 (td, J=1.3, 7.8 Hz, 1H), 6.77 (d,J=5.8 Hz, 1H); LC-MS (ESI+) m/z 231.05 (M+H)⁺; HRMS (ESI+) m/zcalculated for C₁₁H₈ClN₄ (M+H)⁺ 231.0432. found 231.0431.

N⁴-(2-(Trifluoromethyl)phenyl)-2-chloropyrimidine-4-amine (5f, method kin FIG. 5A)

To a solution of 2-(trifluoromethyl)aniline (0.570 g, 3.54 mmol) inanhydrous DMF (4 mL), NaH (60% dispersion in mineral oil, 0.384 g, 9.6mmol) was added portion wise at 0° C. under Argon, followed by theaddition of 2,4-dichloropyrimidine (0.528 g, 3.54 mmol). The reactionmixture was slowly warmed to room temperature and stirred for a further24 h. The solvent was removed under reduced pressure to provide ared/orange solid. The residue was slurried with water (30 mL) and solidwas filtered and washed with water (10 mL×2). The dried solid wassuspended in diethyl ether (5 mL) and sonicated, then filtered andwashed with diethyl ether (5 mL), hexane (10 mL) to afford the titlecompound as a yellow solid. The solid was re-dissolved in ethyl acetate,activated charcoal was added and the mixture was stirred for 20 min atroom temperature. The activated charcoal was removed by filtrationthrough a celite/silica bed. The filtrate was collected, the solventremoved under reduced pressure to provide 5f (0.261 g, 28%) as anoff-white solid. m.p. 139° C. (dec.). ¹H NMR (400 MHz, DMSO-d₆) δ 9.69(s, 1H), 8.11 (d, J=5.9 Hz, 1H), 7.81 (d, J=8.2 Hz, 1H), 7.74 (t, J=7.7Hz, 1H), 7.57-7.51 (m, 2H), 6.57 (d, J=5.9 Hz, 1H); LC-MS (ESI+) m/z274.04 (M+H)⁺; HRMS (ESI+) m/z calculated for C₁₁H₈F₃ClN₃ (M+H)⁺274.0353. found 274.0354.

N⁴-(2-Bromophenyl)-2-chloropyrimidine-4-amine (5g, method b in FIG. 5A)

A solution of 2-bromoaniline (0.200 g, 1.16 mmol) and2,4-dichloropyrimidine (0.175 g, 1.18 mmol) in aqueous HCl (0.1 M, 2.5mL) was stirred at room temperature for 48 h. The precipitate wasfiltered, washed with water (5 mL×2). The dried solid was suspended inan aqueous solution of NaHCO₃ (sat., 20 mL) and extracted with ethylacetate (20 ml×2). The organic extracts were combined, dried overNa₂CO₃, filtered, and the solvent removed under reduced pressure toprovide the title compound 5g (0.207 g, 63%) as an off-white solid andwas used in the next step without further purification. ¹H NMR (400 MHz,DMSO-d₆) 9.70 (s, 1H), 8.11 (d, J=5.8 Hz, 1H), 7.71 (dd, J=1.5, 7.8 Hz,1H), 7.51 (dd, J=1.5, 7.8 Hz, 1H), 7.41 (td, J=1.5, 7.8 Hz, 1H), 7.21(td, J=1.5, 7.8 Hz, 1H), 6.57 (d, J'² 5.8 Hz, 1H); LC-MS (ESI+) m/z285.95 (M+H)⁺; HRMS (ESI+) m/z calculated for C₁₀H₈BrClN₃ (M+H)⁺283.9585. found 283.9586.

N⁴-(2-Iodophenyl)-2-chloropyrimidine-4-amine (5h, method b in FIG. 5A)

A solution of 2-iodoaniline (0.375 g, 1.71 mmol) and2,4-dichloropyrimidine (0.256 g, 1.72 mmol) in aqueous HCl (0.1 M, 3.0mL) was stirred at room temperature for 5 days. The solid precipitatewas filtered and washed with water (30 mL). The dried solid wassuspended in an aqueous solution of NaHCO₃ (sat., 20 mL) and extractedwith ethyl acetate (20 mL×2). The organic extracts were combined, driedover Na₂CO₃, filtered, and the solvent removed under reduced pressure toprovide 5h (0.406 g, 79%) as an off-white solid and was used in the nextstep without further purification. ¹H NMR (400 MHz, DMSO-d₆) 9.68 (s,1H), 8.08 (d, J=5.9 Hz, 1H), 7.93 (dd, J=1.3, 7.9 Hz, 1H), 7.45-7.37 (m,2H), 7.07-7.03 (m, 1H), 6.45 (d, J=5.9 Hz, 1H).

N⁴-(2-Biphenyl)-2-chloropyrimidine-4-amine (5i, method I in FIG. 5A)

This was prepared using 2,4-dichloropyrimidine (0.238 g, 1.597 mmol),biphenyl-2-amine (0.284 g, 1.678 mmol) and DIPEA (0.305 mL, 1.75 mmol)to afford the title compound 5i (0.252 g, 54%) as an off-white solid.r.p. 137.7-138.9° C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.57 (s, 1H), 7.93 (d,J=5.9 Hz, 1H), 7.49-7.19 (m, 9H), 6.32 (br s, 1H); LC-MS (ESI−) m/z282.08 (M+H)⁺; HRMS (ESI+) m/z calculated for C₁₆H₁₃ClN₃ (M+H)⁺282.0793. found 282.0794.

N⁴-(2-Chlorophenyl)-N-methyl-2-chloropyrimidine-4-amine (5j, FIG. 5A)

Iodomethane (0.119 g, 0.837 mmol) was added to a mixture of 21 (0.201 g,0.837 mmol) and cesium carbonate (0.540 g. 1.7 mmol) in anhydrousacetonitrile (1.4 mL) under argon at room temperature. The reactionmixture was then stirred at room temperature overnight. The resultingsolution was diluted with ethyl acetate and the organic layer was washedwith water, separated, dried (Na₂SO₄) and the solvent removed underreduced pressure to provide 5j (0.180 g, 84%) as a yellow oil. Thecompound was used in the next step without further purification. ¹H NMR(400 MHz, CDCl₃) δ 7.90 (d, J=5.3 Hz, 1H), 7.57-7.54 (m, 1H), 7.42-7.35(m, 2H), 7.29-7.27 (m, 1H), 5.83 (s, 1H), 3.43 (s, 3H); LC-MS (ESI+) m/z254.02 (M+H)⁺; HRMS (ESI+) m/z calculated for C₁₁H₁₀Cl₂N₃ (M+H)⁺254.0246. found 254.0252.

N⁴-(2-Chlorophenyl)-N⁴-ethyl-2-chloropyrimidine-4-amine (5k, FIG. 5A)

Iodoethane (0.203 g, 1.30 mmol) was added to a mixture of 21 (0.317 g,1.32 mmol) and cesium carbonate (0.850 g, 2.61 mmol) in anhydrousacetonitrile (2.2 mL) at room temperature overnight under argon. Thereaction mixture was stirred at room temperature overnight under argonovernight. The resulting solution was diluted with ethyl acetate (50 mL)and the organic layer was washed with water, separated, dried (Na₂SO₄)and the solvent removed under reduced pressure to provide a brown oil.Chromatography on silica gel [FlashMaster 3 purification station(hexane:ethyl acetate)] afforded 5k (0.207 g, 71%). as a yellow oil. ¹HNMR (400 MHz, CDCl₃) ¹H NMR (400 MHz, CDCl₃) δ 7.87 (d, J=5.7 Hz, 1H),7.56-7.53 (m, 1H), 7.41-7.34 (m, 2H,), 7.26-7.23 (n, 1H), 5.75 (s, 1H),4.20-4.15 (m, 1H), 3.75 (sextuplet, J=7.1 Hz, 1H), 1.20 (t, J=7.2 Hz,3H); LC-MS (ESI+) m/z 268.03 (M+H)⁺; HRMS (ESI+) m/z calculated forC₁₂H₁₂C₁₂N₃ (M+H)⁺ 268.0403. found 268.0408.

Synthetic protocols for 7a to 7c:

N-(2-Morpholinoethyl)-4-nitrobenzamide (7a, FIG. 6)

2-Morpholinoethylamine (2.1 g, 16.13 mmol) was added to a solution of4-nitrobenzoyl chloride (2.994 g, 16.13 mmol) and triethylamine (2.4 mL)in anhydrous dichloromethane (75 mL) under argon at room temperature.The reaction mixture was stirred under argon at room temperature for twodays. Upon completion of the reaction, the organic phase was washed witha saturated solution of sodium bicarbonate, brine, separated, dried(Na₂SO₄), filtered and the solvent removed under reduced pressure toprovide the title compound 7a (4.082 g, 95%) as a pale brown solid ¹HNMR (400 MHz, DMSO-d₆) δ 8.76 (t, J=5.6 Hz, 1H), 8.29 (d, J=8.7 Hz, 2H),8.03 (d, J=8.7 Hz, 2H), 3.57-3.51 (m, 4H), 3.43-3.36 (m, 2H), 2.50-2.43(m, 2H), 2.42-2.35 (m, 4H); LC-MS (ESI+) m/z 238.2 (M+H)⁺.

N-(2-(Dimethylamino)ethyl)-4-nitrobenzamide (7b, FIG. 6)

This was obtained as a yellow solid (3.770 g, 15.9 mmol, 64%) from4-nitrobenzoyl chloride (3.953 g, 24.9 mmol),N¹,N¹-dimethylethane-1,2-diamine (2.197 g, 24.9 mmol), anhydroustriethylamine (3.75 mL) in the same manner as described for 7a. ¹H NMR(400 MHz, DMSO-d₆) δ 8.80 (t, J=4.9 Hz, 1H), 8.29 (d, J=8.9 Hz, 2H),8.04 (d, J=8.9 Hz, 2H), 3.40 (q, J=6.3 Hz, 2H), 2.49 (m, 2H), 2.23 (s,6H); LC-MS (ESI+) m/z 238.2 (M+H)⁺.

N-(2-Methoxyethyl)-4-nitrobenzamide (7c, FIG. 6)

This was obtained as an off-white solid (3.401 g, 81%) from4-nitrobenzoyl chloride (3.173 g, 20 mmol), 2-methoxyethylamine (1.502g, 20 mmol) and anhydrous triethylamine (3.00 mL) in the same manner asdescribed for 7a. m.p. 108.0-110.5° C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.86(t, J=5.0 Hz, 1H), 8.29 (d, J=8.9 Hz, 2H), 8.05 (d, J=8.9 Hz, 2H),3.50-3.39 (m, 4H), 3.25 (s, 3H). LC-MS (ESI+) m/z 247.1 (M+Na)⁺

Synthetic protocols for 8a to 8c:

4-Amino-N-(2-morpholinoethyl)benzamide (8a, FIG. 6)

The nitrobenzenamide 7a (0.330 g, 1.32 mmol) was dissolved in methanol(15 mL) and hydrogenated using an H-Cube reactor (10% Pd/C, 30 bar, flowrate 1 mL/min, 2 loops, room temperature). The solution was collectedand the solvent removed under reduced pressure to afford 8a (0.330 g,100%) as a pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (t, J=4.9Hz, 1H), 7.52 (d, J=8.6 Hz, 2H), 6.50 (d, J=8.7 Hz, 2H), 5.58 (s, 2H),3.54 (d, J=4.4 Hz, 4H), 3.38-3.23 (m, 2H), 2.50-2.47 (m, 2H), 2.42-2.33(m, 4H); LC-MS (ESI+) m/z 250.2 (M+H)⁺.

4-Amino-N-(2-(dimethylamino)ethyl)benzamide (8b, FIG. 6)

This was obtained as a brown oil (0.174 g, 0.84 mmol) from 7c (0.184 g,0.77 mmol) in the same manner as described for 8a (H-Cube reactor, 10%Pd/C, 20 bar, 1 mL/min, 2 loops, room temperature). The compound wasused in the next step without further purification. ¹H NMR (400 MHz,DMSO-d₆) δ 7.87 (t, J=5.64, 1H), 7.52 (d, J=8.7 Hz, 2H), 6.50 (d, J=8.7Hz, 2H), 5.57 (s, 2H), 3.26 (q, J=6.7 Hz, 2H), 2.34 (t, J=6.9 Hz, 2H),2.14 (s, 6H). LC-MS (ESI+) m/z 208.2 (M+H)⁺.

4-Amino-N-(2-methoxyethyl)benzamide (8c, FIG. 6)

A solution of 7c (0.884 g, 4.2 mmol) in ethanol (20 mL) was stirred inpresence of 10% Pd/C (0.111 g) under hydrogen (balloon) at roomtemperature overnight. The solution was then filtered through celite.The celite was washed with methanol (60 mL). The filtrates were combinedand the solvent removed under reduced pressure to provide 8c (0.730 g,96%) as a yellow oil. ¹H NMR (400 MHz, DMSO-d₆) δ 8.00 (t, J=5.4 Hz,1H), 7.54 (d, J=8.6 Hz, 2H), 6.50 (d, J=8.6 Hz, 2H), 5.58 (s, 1H),3.46-3.29 (m, 4H), 3.23 (s, 3H); LC-MS (ESI+) m/z 195.2 (M+H)⁺

Synthetic Protocols for 10a, 10b, 11a and 11b:

5-(4-Nitrophenyl)-1-tetrazole (10a, FIG. 8)

A solution of 4-nitrobenzonitrile (1.100 g, 7.4 mmol), NaN₃ (1.32 g, 20mmol) and triethylamine hydrochloride (2.73 g, 20 mmol) in toluene (20ml) were heated at 100° C. overnight. (formation of two phases wasobserved). Water (50 ml) was added and the phases were separated. Theaqueous phase was acidified with concentrated HCl (pH=5-6) the solidthat precipitated was filtered and washed with water. The product wasdried under vacuum to give pure 10a (1.30 g, 92%) as a yellow-whitecompound. ¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (d, J=8.8 Hz, 2H), 8.26 (d,J=8.8 Hz, 2H). LC-MS (ESI−) m/z 190.04 (M−H)⁻; HRMS (ESI−) m/zcalculated for C₇H₅N₅O₂ (M−H)⁻ 190.0371. found 190.0367.

5-Nitro-2-(1H-tetrazol-5-yl)phenol (10b, FIG. 8)

A suspension of 2-hydroxy-4-nitrobenzonitrile (1.641 g, 10 mmol), sodiumazide (1.951 g, 30 mmol) and triethylamine hydrochloride (4.130 g, 30mmol) in toluene (20 mL) was stirred at 100° C. for 6 hours. Formationof a biphasic (the upper layer was pale yellow and the lower was winered with the insoluble material) mixture was observed. The mixture wascooled to room temperature and the lower layer solidified. Water (40 ml)was added and the mixture was transferred to a separation funnel. Thered aqueous phase was separated and the organic phase was washed withwater (20 ml×2). The aqueous phase was combined and acidified (conc. HClto pH 5˜6) at 0° C. The precipitate was filtered, washed with water anddried affording the product 10b (1.971, 95%) as an off-white solid. ¹HNMR (400 MHz, DMSO-d₆) δ 8.26-8.20 (m, 1H), 7.83-7.80 (m, 2H); LC-MS(ESI−) m/z 206.04 (M−H)⁻; HRMS (ESI−) m/z calculated for C₇H₄N₅O₃ (M−H)⁻206.0320. found 206.0309.

4-(1-Tetrazol-5-yl)aniline (11a, FIG. 8)

To a solution of 10a (1.1 g, 5.8 mmol) in methanol (70 ml) was added 10%Pd/C (0.25 g). The mixture was degassed and charged with hydrogen(repeated twice) and then stirred under hydrogen balloon overnight(approximately 14 h). The mixture was filtered through a celite pad,washed with methanol, and evaporated to obtain 10a (0.87 g, 93%) as apink-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 7.65 (d, J=8.7 Hz, 2H),6.65 (d, J=8.7 Hz, 2H), 5.76 (s, 1H). LC-MS (ESI−) m/z 160.06 (M−H)⁻;HRMS (ESI−) m/z calculated for C₇H₇N₅ (M−H)⁻ 160.0629. found 160.0625.

5-Amino-2-(1H-tetrazol-5-yl)phenol (11b, FIG. 8)

Nitrotetrazole 10b (0.622 g, 3 mmol) was suspended in methanol (60 ml)and hydrogenated under H₂ atmosphere with Pd/C (10%, 0.1 g) at roomtemperature for 12 hours. The mixture was filtered using a pad of celiteand washed with methanol and the filtrate was concentrated to drynessaffording the product 11b (0.518 g, 98%). as pale yellow solid. ¹H NMR(400 MHz, DMSO-d₆) δ 10.46 (brs, 1H, disappear at D₂O shake), 7.63-7.58(m, 1H), 6.17-6.15 (m, 2H), 5.69 (brs, 2H, disappear at D₂O shake);LC-MS (ESI+) m/z 178.07 (M+H)⁺; HRMS (ESI+) m/z calculated for C₇H₈N₅O(M+H)⁺ 178.0723. found 178.0725.

N⁴-(2-Carboxyphenyl)-N²-(4-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (1, method h in FIG. 4A)

A suspension of 3w (0.040 mg, 0.100 mmol) in NaOH (0.2 mL, 4 M) and THF(0.5 mL) was refluxed in seal tube at 85° C. for 2 h. Upon cooling, THFwas removed and water (2 mL) was added to the mixture, followed byadding HCl (1M) to acidify (pH=2) the mixture. The solid obtained wasfiltered and washed with water (3 mL×2) and MeOH (3 mL×2) and driedunder high vacuum to afford 1 as a white solid (0.033 mg, 85%). m.p.256-258° C. HPLC 98% (R_(t)=7.80 min., 45% MeOH in 0.1% TFA water, 20min.); ¹H NMR (400 MHz, DMSO-d₆) δ 12.57 (br s, 1H), 10.68 (s, 1H), 9.93(s, 1H), 8.44 (appt, J=5.6 Hz, 1H), 8.15 (d, J=6.0 Hz, 1H), 7.98 (dd,J=8.0 Hz, 1H), 7.84-7.79 (m, 4H), 7.60 (t, J=7.6 Hz, 1H), 7.17 (t, J=7.6Hz, 1H), 6.46 (d, J=5.6 Hz, 1H); ¹³C NMR (150 MHz, DMSO-d₆) δ 169.85,167.77, 160.93, 158.28, 155.08, 144.94, 141.45, 134.18, 131.90, 130.81,123.92, 123.22, 122.95, 119.22, 118.83, 101.18; LRMS (ESI−) m/z 351.11(M−Cl)⁺; HRMS (ESI+) m/z calculated for C₁₈H₁₅N₄O₄ (M−Cl)⁺ 351.1088.found 351.1092.

N⁴-(2-Carboxyphenyl)-N²-(4-carbamoylphenyl)pyrimidine-2,4-diaminehydrochloride (3a, method d in FIG. 4A)

A mixture of 2a (0.050 g, 0.175 mmol) and 2-aminobenzamide (0.027 g,0.199 mmol) in HCl (1.0 mL, 0.1 M) was heated in a microwave reactor at160° C. for 15 min. The precipitate obtained upon cooling the mixturewas filtered and washed with water (5 mL) and acetone (5 mL×2) to obtainpure compound 3a (0.036 g, 53%) as a light yellow solid. m.p. 226° C.(dec.). HPLC 96% (R_(t)=3.55 min., 55% MeOH in 0.1% TFA water, 20 min.);¹H NMR (400 MHz, DMSO-d₆) δ 8.82 (d, J=7.6 Hz, 1H), 8.63 (appd, J=7.6Hz, 1H), 8.17 (d, J=8.0 Hz, 1H), 8.03 (dd, J=8.0, 1.4 Hz, 1H), 7.84 (td,J=7.6, 1.2 Hz, 1H), 7.72-7.69 (m, 1H), 7.59 (d, J=8.4 Hz, 1H), 7.40 (t,J=7.6 Hz, 1H), 7.30 (t, J=7.6 Hz, 1H), 6.83 (d, J=8.0 Hz, 1H); LRMS(ESI+) m/z 333.1 (M−NH₂−HCl)⁺; HRMS (ESI+) m/z calculated for C₁₈H₁₅N₅O₂(M−NH₂−HCl)⁺ 333.0982. found 333.1002.

N⁴-(2-Carboxyphenyl)-N²-(phenyl)pyrimidine-2,4-diamine hydrochloride(3b, method d in FIG. 4A)

A mixture of 2a (0.100 g, 0.349 mmol) and aniline (0.038 g, 0.409 mmol)in HCl (1.0 mL, 0.1 M) was heated in a microwave reactor at 160° C. for15 min. The precipitate formed upon cooling the mixture was filtered andwashed with water (5 mL) and hot methanol (5 mL×2) to obtain 3b (0.065g, 54%) as a white solid. m.p. 233° C. (dec.). HPLC 91% (R_(t)=17.27min., 50% MeOH in 0.1% TFA water, 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ10.67 (s, 1H), 9.34 (s, 1H), 8.65 (d, J=8.0 Hz, 1H), 8.11 (d, J=5.6 Hz,1H), 7.97 (d, J=7.6 Hz, 1H), 7.70 (d, J=8.4 Hz, 2H), 7.55 (t, J=6.8 Hz,1H), 7.25 (t, J=8.0 Hz, 2H), 7.08 (t, J=7.6 Hz, 1H), 6.93 (t, J=6.8 Hz,1H), 6.35 (d, J=5.6 Hz, 1H); LRMS (ESI−) m/z 305.1 (M−H−HCl)⁻; HRMS(ESI+) m/z calculated for C₁₇H₁₅N₄O₂ (M−Cl)⁻ 307.1189. found 307.1187.

N⁴-(2-Carboxyphenyl)-N-(4-morpholinophenyl)pyrimidine-2,4-diaminehydrochloride (3c, method e in FIG. 4A)

A mixture of 2a (0.125 g, 0.437 mmol) and 4-morpholinoaniline (0.089 g,0.500 mmol) in a solution of EtOH/1 M HCl (1:1, 2 mL) was heated in amicrowave reactor at 160° C. for 20 min. Addition of EtOAc (0.5 mL) gavea precipitate. The precipitate was filtered and dried under vacuum toafford the desired compound 3c (0.060 g, 32%) as a light yellow solid.m.p. 162° C. (dec.). HPLC 97% (R_(t)=2.99 min., 50% MeOH in 0.1% DEA inwater, 20 min.); ¹H NMR (400 MHz, CD₃OD) δ 8.22 (brs, 1H), 8.14 (dd,J=8.0, 1.6 Hz, 1H), 7.88 (d, J=7.2 Hz, 1H), 7.64-7.54 (m, 5H), 7.35 (t,J=7.6 Hz, 1H), 6.56 (d, J=7.2 Hz, 1H), 4.06 (appt, J=4.6 Hz, 4H), 3.14(appt, J=4.7 Hz, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.36, 162.30,152.53, 144.03, 137.78, 133.70, 131.71, 126.68, 126.62, 124.49, 123.42,119.53, 100.29, 65.36, 52.16; LC-MS (ESI−) m/z 390.15 (M−HCl)⁻; HRMS(ESI−) m/z calculated for C₂₁H₂₀N₅O₃ (M−H−Cl)⁻ 390.1572. found 390.1577.

N⁴-(2-Carboxyphenyl)-N²-(2-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (3d, method d in FIG. 4A)

A suspension of 2,4-dichloropyrimidine (0.149 g, 1.00 mmol) and2-aminobenzoic acid (0.274 g, 2.00 mmol) in HCl (3.0 ml, 0.1 M) washeated in a microwave reactor at 160° C. for 15 min. The precipitateobtained was filtered and washed with water (10 mL), followed by washingwith acetone (5 mL×2) to obtain the desired compound 3d (0.232 g, 60%)as a white solid. m.p. 254° C. (dec.). HPLC 99% (R_(t)=3.53 min., 55%MeOH in 0.1% TFA water, 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 11.80 (s,1H), 8.91 (d, J=8.0 Hz, 1H), 8.22 (dd, J=8.0, 1.2 Hz, 1H), 8.04 (dd,J=8.0, 1.2 Hz, 1H), 7.97-7.93 (m, 2H), 7.76 (td, J=7.6, 1.6 Hz, 1H),7.60 (d, J=8.0 Hz, 1H), 7.54-7.47 (m, 2H), 7.11 (appd, J=8.0 Hz, 1H);LRMS (ESI−) m/z 331.0 (M−OH−HCl)⁻; HRMS (ESI+) m/z calculated forC₁₈H₁₃N₄O₃ (M−HO−HCl)⁺333.0982. found 333.0997.

N⁴-(2-Carboxymethylphenyl)-N²-(4-carboxymethylphenyl)pyrimidine-2,4-diaminehydrochloride (3e, method d in FIG. 4A)

To a mixture of methyl 4-aminobenzoate (0.166 g, 1.099 mmol) and 2e(0.263 g, 0.876 mmol) was added HCl (3.0 mL, 0.1 M). The reactionmixture was heated in a microwave reactor at 160° C. for 15 min. Thecrude product that was precipitated was filtered, dried under vacuum andpurified using SiO₂ chromatography (gradient elution 0-20% EtOAc inhexane) to obtain the desired product 3e (0.116 g, 32%) as a whitesolid. m.p. 196-198° C. HPLC 99% (R_(t)=5.82 min., 60% MeOH in 0.1% TFAwater, 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 10.08 (s, 1H), 9.73 (s,1H), 8.37 (d, J=8.4 Hz, 1H), 8.16 (d, J=6.0 Hz, 1H), 7.94 (dd, J=8.0,1.6 Hz, 1H), 7.84 (d, J=9.2 Hz, 2H), 7.80 (d, J=9.2 Hz, 2H), 7.64-7.60(m, 1H), 7.18 (appt, J=7.2 Hz, 1H), 6.45 (d, J=6.0 Hz, 1H), 3.81 (s,3H), 3.79 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.25, 166.69, 160.74,159.58, 157.54, 146.04, 141.41, 134.30, 131.39, 130.62, 123.02, 122.94,121.99, 118.58, 118.39, 101.12, 52.97, 52.35; LRMS (ESI+) m/z 379.1(M−Cl)⁺; HRMS (ESI+) m/z calculated for C₂₀H₁₉N₄O₄ (M−Cl)⁺ 379.1401.found 379.1402.

N⁴,N²-Diphenylpyrimidine-2,4-diamine hydrochloride (3f, method d in FIG.4A)

A mixture of 2,4-dichloropyrimidine (0.050 g, 0.336 mmol) and aniline(0.063 g, 0.677 mmol) in HCl (1.0 mL, 0.1 M) was heated in a microwavereactor at 160° C. for 15 min. The product obtained was purified usingSiO₂ chromatography (gradient elution 0-20% EtOAc in hexane) to affordthe desired product 3f (0.048 g, 49%) as a white solid. m.p. 144° C.(dec.). HPLC 96% (R_(t)=5.33 min., 60% MeOH in 0.1% TFA water, 40 min.);¹H NMR (400 MHz, CD₃OD) δ 7.85 (d, J=6.0 Hz, 1H), 7.59 (d, J=7.6 Hz,2H), 7.55 (dd, J=7.6 Hz, 2H), 7.27 (d, J=7.6 Hz, 2H), 7.23 (d, J=7.6 Hz,2H), 7.05-6.98 (m, 2H), 6.20 (d, J=6.0 Hz, 1H); LRMS (ESI+) m/z 263.1(M−Cl)⁺; HRMS (ESI+) m/z calculated for C₁₆H₁₅N₄ (M−Cl)⁺ 263.1291. found263.1293.

N⁴-(2-Carbamoylphenyl)-N²-(4-carbamoyl)pyrimidine-2,4-diaminehydrochloride (3g, method din FIG. 4A)

A mixture of 2b (0.248 g, 0.871 mmol) and 4-aminobenzamide (0.136 g,1.00 mmol) in HCl (3.0 mL, 0.1 M) was heated in a microwave reactor at160° C. for 15 min. The precipitate was filtered and washed with water(10 mL), hot MeOH (10 mL), hot THF (10 mL), dioxane (5 mL), DMF (5 mL)and MeOH (10 mL) sequentially to obtain pure 3g (0.160 g, 48%) as alight yellow solid. m.p. 257-260° C. HPLC 97% (R_(t)=3.32 min., 50% MeOHin 0.1% TFA water, 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ10.63 (s, 1Hdisappeared on D₂O shake), 9.65 (s, 1H disappeared on D₂O shake), 8.57(d, J=8.4 Hz, 1H), 8.15 (d, J=5.2 Hz, 1H), 7.98 (dd, J=7.6, 1.6 Hz, 1H),7.83-7.73 (m, 5H becomes 4H on D₂O shake), 7.63-7.58 (m, 1H), 7.17 (brs,1H disappeared on D₂O shake), 7.12 (t, J=8.4 Hz, 1H), 6.42 (d, J=6.0 Hz,1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.31, 168.30, 160.55, 159.67,157.36, 144.07, 142.41, 134.35, 131.96, 128.81, 127.22, 122.25, 121.83,118.50, 117.57, 101.05; LRMS (ESI+) m/z 349.1 (M−Cl)+; HRMS (ESI+) m/zcalculated for C₁₈H₁₇N₆O₂ (M−Cl)⁺ 349.1408. found 349.1407.

N⁴-(Phenyl)-N²-(4-carboxyphenyl)pyrimidine-2,4-diamine hydrochloride(3h, method d in FIG. 4A)

A mixture of 2c (0.020 g, 0.082 mmol) and 4-aminobenzoic acid (0.013 g,0.097 mmol) in HCl (1.5 mL, 0.1 M) was heated in a microwave reactor at160° C. for 15 min. The solid obtained upon cooling the mixture wasfiltered, washed with water (5 mL) and dried under vacuum to obtain thedesired compound 3h (0.020 g, 71%) as a white solid. m.p. 215° C.(dec.). HPLC 99% (R_(t)=7.28 min., 50% MeOH in 0.1% TFA water, 20 min.);¹H NMR (400 MHz, CD₃OD) δ 8.00 (d, J=8.8 Hz, 2H), 7.86 (d, J=7.2 Hz,1H), 7.63 (d, J=8.4 Hz, 2H), 7.57 (d, J=7.6 Hz, 2H), 7.41 (t, J=7.6 Hz,2H), 7.28 (t, J=7.6 Hz, 1H), 6.46 (d, J=7.2 Hz, 1H); ¹³C NMR (100 MHz,DMSO-d₆) 167.44, 161.79, 152.70, 144.17, 141.82, 137.81, 130.87, 129.50,126.80, 126.15, 123.30, 121.17, 100.58. LRMS (ESI−) m/z 305.0 (M−Cl)⁻;HRMS (ESI+) m/z calculated for C₁₇H₁₅N₄O₂ (M−Cl)⁺ 307.1190. found307.1187.

N⁴,N²-Di(4-carboxyphenyl)pyrimidine-2,4-diamine hydrochloride (3i,method d in FIG. 4A)

A mixture of 2d (0.100 g, 0.350 mmol) and 4-aminobenzoic acid (0.055 g,0.401 mmol) in HCl (1.5 mL, 0.1 M) was heated in a microwave reactor at160° C. for 15 min. The precipitate obtained upon cooling the mixturewas filtered and washed with water (10 mL), followed by quick wash withacetone (5 mL×2) to give pure 3i (0.136 g, 99%) as a white solid. m.p.281-283° C. HPLC 98% (R_(t)=4.97 min., 50% MeOH in 0.1% TFA in water, 20min.); ¹H NMR (400 MHz, DMSO-d₆) δ 10.71 (brs, 1H), 10.46 (brs, 1H),8.09 (d, J=6.8 Hz), 7.90 (d, J=6.0 Hz, 2H partially overlapping) 7.88(d, J=6.0 Hz, 2H partially overlapping), 7.79 (d, J=8.8 Hz, 2H), 7.71(d, J=8.4 Hz, 2H), 6.50 (d, J=6.4 Hz, 1H); LRMS (ESI−) m/z 349.0(M−H−HCl)⁻; HRMS (ESI+) m/z calculated for C₁₈H₁₅N₄O₄ (M−Cl)⁺ 351.1088.found 351.1082.

N⁴-(2-Carboxyphenyl)-N²-(4-carboxyphenyl)-5-methylpyrimidine-2,4-diaminehydrochloride (3j, method g in FIG. 4A)

A mixture of 2j (0.115 g, 0.383 mmol) and 4-aminobenzoic acid (0.179 g,1.307 mmol) in HCl (1 M)/THF (2:1, 3.0 mL) was heated in a microwavereactor at 160° C. for 15 min. The precipitate obtained upon cooling themixture was filtered and washed with water (5 mL) and MeOH (5 mL×2). Thesolid obtained was slurried in DMF (2 mL), filtered and washed with MeOH(5 mL), acetone (5 mL) and DCM (5 mL) sequentially to obtain the desiredcompound 3j (0.055 g, 36%) as a white solid. m.p. 213° C. (dec.). HPLC97% (R_(t)=5.17 min., 55% MeOH in 0.1% TFA water, 20 min.); ¹H NMR (400MHz, DMSO-d₆) δ 12.50 (brs, 1H, disappeared on D₂O shake), 11.00 (s, 1Hdisappear on D₂O shake), 9.68 (s, 1H disappeared on D₂O shake), 9.01 (d,J=8.4 Hz, 1H), 8.08 (s, 1H), 8.03 (d, J=7.9 Hz, 1H), 7.85-7.80 (m, 4H),7.60 (t, J=7.9 Hz, 1H), 7.11 (t, J=7.6 Hz, 1H), 2.16 (s, 3H); LC-MS(ESI+) m/z 365.13 (M−Cl)⁺; HRMS (ESI+) m/z calculated for C₁₉H₁₇N₄O₄(M−Cl)⁺ 365.1244. found 365.1240.

N⁴-(2-Carboxyphenyl)-N-(4-carboxyphenyl)-6-methylpyrimidine-2,4-diaminehydrochloride (3k, method d in FIG. 4A)

A mixture of 2f (0.263 g, 0.877 mmol) and 4-aminobenzoic acid (0.137 g,1.00 mmol) in HCl (3.0 mL, 0.1 M) was heated in a microwave reactor at160° C. for 15 min. The precipitate obtained was filtered and washedwith water (10 mL), MeOH (10 mL×2), DMSO (1 mL, quick wash), and acetone(5 ml) to obtain the desired compound 3k (0.187 g, 53%) as a whitesolid. m.p. 243° C. (dec.). HPLC 90% (R_(t)=5.11 min., 55% MeOH in 0.1%TFA water, 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 10.56 (s, 1H), 9.73 (s,1H), 8.55 (d, J=8.4 Hz, 1H), 7.98 (d, J=6.8 Hz, 1H), 7.86 (d, J=8.8 Hz,2H), 7.81 (d, J=8.8 Hz, 2H), 7.58 (t, J=7.2 Hz, 1H), 7.11 (t, J=7.2 Hz,1H), 6.33 (s, 1H), 2.28 (s, 3H); LC-MS (ESI−) m/z 363.11 (M−H−HCl)⁻;HRMS (ESI−) m/z calculated for C₁₉H₁₅N₄O₄ (M−H−HCl)⁻ 363.1099. found363.1107.

N⁴-(2-Chlorophenyl)-N²-(4-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (3l, method e in FIG. 4A)

A mixture of 21 (0.094 g, 0.340 mmol) and 4-aminobenzoic acid (0.107 g,0.781 mmol) in EtOH/1M HCl (2.0 mL, 1:1) was heated in a microwavereactor at 160° C. for 15 min. The mixture was cooled to r.t. Theprecipitate obtained was filtered, washed with water (2 mL), and MeOH (2mL) sequentially to afford the desired compound 3l (0.055 g, 43%) as awhite solid. m.p. 234° C. (dec.). HPLC 99% (R_(t)=4.32 min., 55% MeOH in0.1% TFA water, 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 12.59 (s, 1H),10.00 (s, 1H), 9.76 (s, 1H), 8.06 (d, J=6.0 Hz, 1H), 7.70-7.68 (m, 3H),7.63-7.58 (m, 3H), 7.43 (t, J=7.8 Hz, 1H), 7.33 (t, J=7.4 Hz, 1H), 6.40(d, J=6.1 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) 167.36, 163.18, 152.30,145.07, 141.91, 134.61, 130.72, 130.64, 130.47, 129.67, 129.52, 128.56,126.30, 120.01, 95.58; LC-MS (ESI+) m/z 341.09 (M−Cl)⁺; HRMS (ESI+) m/zcalculated for C₁₇H₁₄ClN₄O₂ (M−Cl)⁺ 341.0800. found 341.0810.

N⁴-(2-Carboxyphenyl)-N²-phenyl-5-fluoropyrimidine-2,4-diaminehydrochloride (3m, method e in FIG. 4A)

A mixture of 2g (0.134 g, 0.441 mmol) and aniline (0.140 g, 1.50 mmol)in 1:1 ratio of EtOH/1M HCl (2.0 mL) was heated in a microwave reactorat 160° C. for 30 min. The precipitate formed upon cooling the mixturewas filtered and washed with MeOH (5 mL), acetone (5 mL) and dried undervacuum to afford the desired compound 3m (0.150 g, 94%) as a whitesolid. m.p. 240° C. (dec.). HPLC 99% (R_(t)=4.21 min., 70% MeOH in 0.1%TFA water, 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 11.50 (s, 1H), 9.41 (s,1H), 8.96 (d, J=8.4 Hz, 1H), 8.21 (d, J=3.1 Hz, 1H), 8.04 (dd, J=7.9,1.5 Hz, 1H), 7.67 (d, J=8.4 Hz, 2H), 7.60 (t, J=8.4 Hz, 1H), 7.27 (t,J=8.4 Hz, 2H), 7.12 (t, J=7.6 Hz, 1H), 6.94 (t, J=7.3 Hz, 1H); ¹⁹F NMR(376 MHz, DMSO-d₆) δ −166.03 (s); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.83,156.07 (d, J=3.0 Hz), 149.60 (d, J=10.0 Hz), 142.26, 141.68 (d, J=245Hz), 141.52, 141.33, 141.25, 134.72, 132.02, 129.05, 122.15 (d, J=21.0Hz), 120.74, 119.80, 116.24; LC-MS (ESI+) m/z 325.12 (M−Cl)⁺; HRMS(ESI+) m/z calculated for C₁₇H₁₄FN₄O₂ (M−Cl)⁺ 325.1095. found 325.1093.

N⁴-(2-Carboxyphenyl)-N²-(4-carboxyphenyl)-5-fluoropyrimidine-2,4-diamineacid (3n, method e in FIG. 4A)

A mixture of 2g (0.134 g, 0.44 mmol) and 4-aminobenzoic acid (0.206 g,1.50 mmol) in 1:1 ratio of EtOH/HCl (2.0 mL, 1 M) was heated in amicrowave reactor at 160° C. for 30 min. The precipitate formed uponcooling the mixture was filtered and washed with sat. NaHCO₃ (3 mL) andwater (5 mL). The solid obtained was slurried in hot DMF (3 mL),filtered and washed with MeOH (5 mL) and dried under vacuum to affordthe desired compound 3n (0.096 g, 59%) as a white solid. m.p. 287-290°C. HPLC 99% (R_(t)=14.73 min., 55% MeOH in 0.1% TFA, water 20 min.); ¹HNMR (400 MHz, DMSO-d₆) δ 11.42 (s, 1H disappeared on D₂O shake), 9.83(s, 1H disappeared on D₂O shake), 8.94 (d, J=8.6 Hz, 1H), 8.29 (appd,J=2.7 Hz, 1H), 8.05 (d, J=7.9 Hz, 1H), 7.83 (d, J=8.4 Hz, 2H), 7.83 (d,J=8.4 Hz, 2H), 7.63 (t, J=7.2 Hz, 1H), 7.16 (t, J=7.5 Hz, 1H); ¹⁹F NMR(376 MHz, DMSO-d₆) δ −164.42 (s); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.76,167.82, 155.42 (d, J=3.5 Hz), 149.76 (d, J=10 Hz), 145.58, 142.09 (d,J=247 Hz), 142.00, 141.25 (d, J=18 Hz), 134.70, 132.03, 130.87, 123.39,122.57, 120.93, 118.13, 116.65; LC-MS (ESI+) m/z 369.10 (M+H)⁺; HRMS(ESI+) m/z calculated for C₁₈H₁₄FN₄O₄ (M+H)⁺ 369.0994. found 369.0994.

N⁴-(2-Chlorophenyl)-N²-(4-carboxyphenyl)-5-fluoropyrimidine-2,4-diaminehydrochloride (3o, method d in FIG. 4A)

A mixture of 2k (0.300 g, 1.12 mmol) and 4-aminobenzoic acid (0.153 g,1.12 mmol) in ethanol (1.5 mL) was heated in a microwave reactor at 150°C. for 20 min. The reaction mixture was cooled, and stirred at roomtemperature for 48 hours. The white precipitate was isolated byfiltration and washed with ethyl acetate (5 mL). The product wassuspended in ethyl acetate (5 mL) and sonicated for 5 min. and filteredto provide 3o (0.232 g, 52%) as a white powder. m.p. 308° C. (dec.).HPLC 99% (R_(t)=7.57 min., 55% MeOH in 0.1% TFA water, 20 min.); ¹H NMR(400 MHz, DMSO-d₆) δ 12.44 (s, 1H), 9.59 (s, 1H), 9.38 (s, 1H), 8.15 (d,J=3.4 Hz, 1H), 7.62-7.52 (m, 6H), 7.45-7.36 (m, 2H); ¹⁹F NMR (376 MHz,DMSO-d₆) δ −164.92; ¹³C NMR (100 MHz, DMSO-d₆) δ 167.77, 155.67 (d,J=3.0 Hz), 151.48 (d, J=12.0 Hz), 145.80, 141.60 (d, J=20.0 Hz), 141.46(d, J=245.0 Hz), 136.04, 131.63, 130.55, 130.40, 130.19, 128.44, 128.38,122.66, 117.37; LC-MS (ESI+) m/z 359.07 (M−Cl)⁺; HRMS (ESI+) m/zcalculated for C₁₇H₁₃ClFN₄O₂ (M−Cl)⁺ 359.0706. found 359.0709.

N⁴-(2-Chlorophenyl)-N²-phenyl-5-fluoropyrimidine-2,4-diaminehydrochloride (3p, method m in FIG. 4A)

A mixture of 2k (0.061 g, 0.207 mmol) and aniline (0.020 g, 0.207 mmol)in EtOH (2.0 mL) was heated in a microwave reactor at 150° C. for 20min. The precipitate formed upon cooling the mixture was filtered andquickly washed with MeOH (1 mL) to afford the desired product 3p (0.037g, 51%) as a white solid. m.p. 145-148° C. HPLC 99% (R_(t)=9.43 min.,55% MeOH in 0.1% TFA water, 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 9.18(s, 1H disappeared on D₂O shake), 9.12 (s, 1H disappeared on D₂O shake),8.08 (d, J=4.0 Hz, 1H), 7.59-7.56 (m, 2H), 7.44 (d, J=8.4 Hz, 2H), 7.39(td, J=7.6, 1.6 Hz, 1H), 7.31 (td, J=7.6, 1.6 Hz, 1H), 7.02 (t, J=7.2Hz, 2H), 6.77 (t, J=7.6 Hz, 1H); ¹⁹F NMR (376 MHz, DMSO-d₆) δ −166.49;¹³C NMR (100 MHz, DMSO-d₆) δ 155.97, 151.42 (d, J=12 Hz), 141.40, 141.24(d, J=20 Hz), 141.11 (d, J=244 Hz), 136.05, 131.18, 130.35, 129.80,128.76, 128.29, 128.13, 121.33, 118.80; LC-MS (ESI+) m/z 315.08 (M-Cl)⁺;HRMS (ESI+) m/z calculated for C₁₆H₁₃ClFN₄(M−Cl)⁺ 315.0807. found315.0812.

N⁴-(2-Carboxyphenyl)-N²-(3-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (3q method din FIG. 4A)

A mixture of 2a (0.050 g, 0.175 mmol) and 3-aminobenzoic acid (0.024 g,0.175 mmol) in HCl (0.1 mL, 0.1 M) was heated in a microwave reactor at160° C. for 15 min. The solution was cooled and the precipitate obtainedwas filtered and washed quickly with hot methanol (3 ml) to obtain thedesired product 3q (0.056 g, 72%) as a white solid. m.p. 275° C. (dec).HPLC 99% (R_(t)=6.31 min., 50% MeOH in 0.1% TFA water, 20 min.); ¹H NMR(400 MHz, DMSO-d₆) δ 12.85 (brs, 1H), 10.73 (s, 1H), 9.52 (s, 1H), 8.67(d, J=8.4 Hz, 1H), 8.27 (s, 1H), 8.12 (d, J=5.7 Hz, 1H), 7.96 (d, J=7.9Hz, 2H), 7.54-7.47 (m, 2H), 7.51 (t, J=7.7 Hz, 2H), 7.05 (t, J=7.6 Hz,1H), 6.36 (d, J=5.7 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) 170.49, 168.16,160.41, 159.99, 157.52, 142.75, 141.52, 134.46, 131.93, 131.72, 129.22,124.08, 122.79, 121.79, 121.25, 120.71, 116.79, 100.98. LC-MS (ESI−) m/z349.10 (M−H−HCl)⁻; HRMS (ESI−) m/z calculated for C₁₈H₁₄N₄O₄ (M−H−HCl)⁻349.0942. found 397.0940.

N⁴-(2-Carboxyphenyl)-N²-(3-carboxyphenyl)-5-fluoropyrimidine-2,4-diaminehydrochloride (3r method m in FIG. 4A)

A mixture of 2g (0.100 g, 0.329 mmol) and 3-aminobenzoic acid (0.048 g,0.350 mmol) in HCl (1.0 mL, 0.1 M) was heated in a microwave reactor at150° C. for 20 min. The mixture was cooled to r.t. and the precipitateobtained was filtered and washed quickly with ethanol (3 ml) to obtainthe desired product 3r (0.089 g, 66%) as a white solid. m.p. 272° C.(dec.). HPLC 99% (R_(t)=14.34 min., 50% MeOH in 0.1% TFA water, 20min.); ¹H NMR (400 MHz, DMSO-d₆) δ 13.07 (brs, 1H), 11.48 (s, 1H), 9.61(s, 1H), 8.97 (d, J=8.4 Hz, 1H), 8.31-8.23 (m, 2H), 8.03 (d, J=7.9 Hz,1H), 7.60-7.48 (m, 2H), 7.39 (t, J=7.9 Hz, 1H), 7.12 (t, J=7.6 Hz, 1H);¹³C NMR (101 MHz, DMSO-d₆) δ 170.87, 168.29, 155.90, 149.69 (d, J=9.7Hz), 151.37 (d, J=11.5 Hz), 141.91 (d, J=235.3 Hz), 142.22, 141.54,134.89, 132.04, 131.77, 129.32, 123.78, 122.87, 122.29, 120.62, 120.37,116.19; ¹⁹F NMR (376 MHz, DMSO) δ −165.34; Elemental analysis:calculated for C₁₈H₁₄ClFN₄O₄ C, 53.41; H, 3.49; N, 13.84. Found: C,53.22; H, 3.37; N, 13.57.

N⁴-(2-Carboxyphenyl)-N-(4-ethoxycarbonylphenyl)-5-chloropyrimidine-2,4-diaminehydrochloride (3s, method f (ii) in FIG. 4A)

A mixture of 2m (0.063 g, 0.193 mmol) and ethyl 4-aminobenzoate (0.043g, 0.260 mmol) in ethanol (0.8 mL), was heated in a sealed tube at 120°C. (oil bath temperature) overnight. The resulting precipitate wasfiltered and washed with ethanol (1 mL×2), Et₂O (3 mL), hexane (2 mL)sequentially and dried under vacuum to afford the title compound 3s(0.079 g, 90%), as a white solid. m.p. 221° C. (dec.). ¹H NMR (400 MHz,DMSO-d₆) δ 13.77 (s, 1H), 11.46 (s, 1H), 9.97 (s, 1H), 8.93 (d, J=8.3Hz, 1H), 8.34 (s, 1H), 8.04 (dd, J=1.5, 7.9 Hz, 1H), 7.86 (d, J=9.1 Hz,2H), 7.83 (d, J=9.1 Hz, 2H), 7.64-7.60 (m, 1H), 7.19-7.15 (m, 1H), 4.26(q, J=7.1 Hz, 2H), 1.29 (t, J=7.1 Hz, 3H). LC-MS (ESI−) m/z 412.09(M+H)⁺; HRMS (ESI−) m/z calculated for C₂₀H₁₈ClN₄O₄ (M+H)⁺ 413.1011.found 413.0989.

N⁴-(2-Carboxyphenyl)-N²-(4-ethoxycarbonylphenyl)-5,6-dichloropyrimidine-2,4-diaminehydrochloride (3t, method f (ii) in FIG. 4A)

A mixture of 2n (0.091 g, 0.257 mmol) and ethyl 4-aminobenzoate (0.044g, 0.266 mmol) in ethanol (1.0 mL), was heated in a sealed tube at 110°C. (oil bath temperature) for 4 days. The resulting precipitate wasfiltered and washed with ethanol (1 mL×2) and suspended in methanol (1mL). The mixture was sonicated and the solid was filtered and driedunder vacuum to afford the title compound 3t (0.022 g, 17%) as a whitesolid. m.p. 206° C. (dec.). ¹H NMR (400 MHz, DMSO-d₆) δ 11.52 (s, 1H),10.25 (s, 1H), 8.74 (s, 1H), 8.04 (dd, J=1.5, 7.9 Hz, 1H,), 7.86 (d,J=8.8 Hz, 2H), 7.75 (d, J=8.9 Hz, 2H), 7.64-7.60 (m, 1H,), 7.23-7.20 (m,1H), 4.27 (q, J=7.1 Hz, 2H,), 1.29 (t, J=7.1 Hz, 3H). LC-MS (ESI−) m/z447.07 (M−Cl); HRMS (ESI−) m/z calculated for C₂₀H₁₇Cl₂N₄O₄ (M−Cl)⁺447.0621. found 447.00598.

5-Amino-N⁴-(2-carboxyphenyl)-N²-(4-ethoxycarbonylphenyl)pyrimidine-2,4-diaminehydrochloride (3u, method f (i) in FIG. 4A)

This was prepared by using a method described Gray and co-workers.⁴⁷ Amixture of 2h (0.080 g, 0.266 mmol), ethyl 4-aminobenzoate (0.099 g,0.600 mmol) and HCl (0.15 mL, 4M in dioxane) in 2-butanol (1.0 mL) washeated in a sealed tube at 120° C. (oil bath temperature) for 24 h.After cooling the mixture to r.t., the suspension obtained was filteredand washed with water (5 mL), MeOH (3 mL) to afford the desired compound3u (0.082 g, 72%) as a yellow solid. m.p. 262° C. (dec.). HPLC 99%(R_(t)=15.48 min., 50% MeOH in 0.1% TFA water, 20 min.); ¹H NMR (400MHz, DMSO-d₆) δ 9.88 (s, 1H), 9.87 (s, 1H), 9.18 (s, 1H), 7.94-7.84 (m,5H), 7.80 (d, J=8.0 Hz, 1H), 7.42 (t, J=8.4 Hz, 1H), 7.19 (d, J=8.0 Hz,1H), 7.04 (t, J=7.5 Hz, 1H), 4.26 (q, J=7.0 Hz, 2H), 1.30 (t, J=7.0 Hz,3H); LC-MS (ESI+) m/z 376.15 (M−OH−HCl)⁺; HRMS (ESI+) m/z calculated forC₂₀H₁₈N₅O₃ (M−OH−HCl)⁺ 376.1404. found 376.1405.

6-Amino-N⁴-(2-carboxyphenyl)-N²-(4-ethoxycarbonylphenyl)pyrimidine-2,4-diaminehydrochloride (3v, method e in FIG. 4A)

A mixture of 2i (0.265 g, 0.880 mmol) and ethyl 4-aminobenzoate (1.65 g,10.00 mmol) in EtOH/1 M HCl (1:1, 12 mL) was heated in a microwavereactor at 160° C. for 1 h. The mixture was cooled to r.t., and thesolid obtained was filtered, washed with MeOH (5 mL) and slurried inacetone (10×5 mL) until no impurity was shown by NMR to afford thedesired compound 3v (0.125 g, 33%) as a beige color solid. m.p. 280° C.(dec.). ¹H NMR (400 MHz, DMSO-d₆) δ 10.65 (s, 1H disappeared on D₂Oshake), 9.36 (s, 1H disappeared on D₂O shake), 8.85 (appd, J=7.6 Hz,1H), 7.94 (d, J=8.4 Hz, 1H), 7.83 (d, J=8.0 Hz, 2H), 7.65 (d, J=8.4 Hz,2H), 7.47 (t, J=8.4 Hz, 1H), 6.94 (t, J=8.0 Hz, 1H), 6.54 (brs, 2Hdisappeared on D₂O shake), 5.54 (s, 1H), 4.26 (q, J=7.0 Hz, 2H), 1.29(t, J=7.0 Hz, 3H); LC-MS (ESI+) m/z 394.15 (M−Cl)⁺; HRMS (ESI+) m/zcalculated for C₂₀H₂₀N₅O₄ (M−Cl)⁻ 394.1510. found 394.1509.

N⁴-(2-Carboxyphenyl)-N²-(4-methoxycarbonylphenyl)pyrimidine-2,4-diaminehydrochloride (3w, method m in FIG. 4A)

A suspension of 2a (0.060 g, 0.210 mmol) and methyl 4-aminobenzoate(0.032 g, 0.210 mmol) in MeOH (1 mL) was heated in a microwave reactorat 150° C. for 20 min. Upon cooling, the resulting precipitate wasfiltered and washed with MeOH (2 mL) to afford 3w (0.064 mg, 76%) as ayellow solid which was used without further purification. ¹H NMR (400MHz, DMSO-d₆) δ 13.40 (br s, 1H), 10.74 (s, 1H), 10.11 (s, 1H), 8.33 (brd, J=5.6 Hz, 1H), 8.14 (d, J=6.0 Hz, 1H), 7.99 (d, J=8.0 Hz, 1H),7.84-7.76 (m, 4H), 7.63 (t, J=7.6 Hz, 1H), 7.22 (t, J=7.6 Hz, 1H), 6.50(d, J=6.4 Hz, 1H), 3.80 (s, 3H); LC-MS (ESI+) m/z 365.13 (M−Cl)⁺; HRMS(ESI+) m/z calculated for C₁₉H₁₇N₄O₄ (M−Cl)⁺ 365.1244. found 365.1243.

4-(4-(2,6-Dichlorophenylamino)-5-fluoropyrimidin-2-ylamino)benzoic acidhydrochloride (3x, method f (ii) in FIG. 4A)

A solution of 2-chloro-N-(2,6-dichlorophenyl)-5-fluoropyrimidin-4-amine(50 mg, 0.17 mmol) and 4-aminobenzoic acid (23 mg, 0.17 mmol) in ethanol(1 ml) were heated at 100° C. for 18 h. The compound precipitated wasfiltered and washed quickly with methanol to provide pure 3× as a whitesolid (36 mg, 49%). ¹H NMR (400 MHz, DMSO) δ 9.92 (s, 1H), 9.85 (s, 1H),8.23 (d, J=3.4 Hz, 1H), 7.67 (d, J=8.0 Hz, 2H), 7.56 (d, J=8.6 Hz, 2H),7.49 (t, J=8.1 Hz, 1H), 7.41 (s, 2H). ¹⁹F NMR (376 MHz, DMSO) δ −164.28(s); LC-MS (ESI−) m/z 391.03 (M−H−HCl)⁻; HRMS (ESI-ve) m/z calculatedfor C₁₇H₁₀Cl₂FN₄O₂ (M−H−HCl)⁻ 391.0170. found 391.0140.

4-(5-Fluoro-4-(2-nitrophenylamino)pyrimidin-2-ylamino)benzoic acidhydrochloride (3y, method f (ii) in FIG. 4A)

A solution of 2-chloro-5-fluoro-N-(2-nitrophenyl)pyrimidin-4-amine (40mg, 0.15 mmol) and 4-aminobenzoic acid (23 mg, 0.17 mmol) in ethanol (1ml) were heated at 100° C. for 44 h. The compound precipitated wasfiltered and washed quickly with methanol to provide pure 3y as a whitesolid (33 mg, 53%); ¹H NMR (400 MHz, DMSO) δ 10.03 (s, 1H), 9.70 (s,1H), 8.28 (d, J=3.3 Hz, 1H), 8.12 (dd, J=8.3, 1.4 Hz, 1H), 8.05 (d,J=8.1 Hz, 1H), 7.86-7.76 (m, 1H), 7.70 (d, J=8.9 Hz, 2H), 7.63 (d, J=8.9Hz, 2H), 7.54-7.35 (m, 1H); LC-MS (ESI−) m/z 368.09 (M−H−HCl)⁻; HRMS(ESI-ve) m/z calculated for C₁₇H₁₁FN₅O₄ (M−H−HCl)⁻ 368.0801. found368.0768.

5-Amino-N⁴-(2-carboxyphenyl)-N²-(4-carboxyphenyl)pyrimidine-2,4-diamine(4a, method h in FIG. 4A)

A suspension of 3u (0.069 g, 0.161 mmol) in NaOH/THF (2 M, 0.44 mL/0.2mL) was heated at 100° C. (oil bath temperature) in a sealed tube for 30min. A solution of HCl (1 M) was added to acidify the solution to pH=1-2after removing THF. The solid obtained was filtered and washed withwater (5 mL), sat. NaHCO₃ (3 mL), water (5 mL), acetone (3 mL), and MeOH(3 mL) sequentially to afford the desired compound 4a (0.038 g, 65%) asa brown solid. m.p. 160° C. (dec.). HPLC 96% (RA=4.19 min., 55% MeOH in0.1% TFA water 20 min.); ¹H NMR (400 MHz, DMSO-do) δ 9.33 (brs, 1Hdisappeared on D₂O shake), 8.86 (brs, 1H), 7.99 (brs, 1H), 7.88-7.78 (m,5H), 7.50 (brs, 1H), 7.01 (brs, 1H); LC-MS (ESI+) m/z 366.12 (M+H)⁺;HRMS (ESI+) m/z calculated for C₁₈H₁₆N₅O₄ (M+H)⁺ 366.1197. found366.1198.

6-Amino-N⁴-(2-carboxyphenyl)-N²-(4-carboxyphenyl)pyrimidine-2,4-diamine(4b, method h in FIG. 4A)

A suspension of 3v (0.070 g, 0.163 mmol) in NaOH (0.45 mL, 2 M) and THF(0.25 mL) was heated at 100° C. (oil bath temperature) in a sealed tubefor 16 h. The THF in the mixture was evaporated and HCl (1 M) was addedto acidify (pH=1-2) the mixture. The solid obtained was filtered andwashed with water (3 mL), sat. aq. NaHCO₃ (3 mL) and water (3 mL)sequentially, and dried to afford the desired compound 4b (0.047 g, 79%)as a yellow solid. m.p. 220° C. (dec.). HPLC 95% (RA=5.20 min., 55% MeOHin 0.1% TFA water, 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 10.65 (s, 1Hdisappeared on D₂O shake), 9.78 (s, 1H disappeared on D₂O shake), 8.54(brs, 1H), 7.96 (appd, J=5.2 Hz, 1H), 7.82-7.80 (m, 2H), 7.59-7.53 (m,3H), 7.34-7.10 (m, 3H), 5.60 (s, 1H); LC-MS (ESI+) 366.13 m/z (M+H)⁺;HRMS (ESI+) m/z calculated for C₁₈H₁₆N₅O₄ (M+H)⁺ 366.1197. found366.1194.

N⁴-(2-Carboxyphenyl)-N-(4-carboxyphenyl)-5,6-dichloropyrimidine-2,4-diaminehydrochloride (4c, method k in FIG. 4A)

A mixture of 3t (0.018 g, 0.034 mmol) in THF (0.3 mL) and NaOH (0.1 mL,2 M) was heated in a sealed tube at 110° C. (oil bath temperature)overnight. The THF was then removed under reduced pressure and HCl (1 Maq., 0.5 mL) was added to the residue. The resulting precipitate wasfiltered and washed with water (2 mL) and dried under vacuum. The solidobtained was then slurried in methanol (1 mL), filtered and dried undervacuum to afford the title compound 4c (0.005 g, 32%) as a white solid.m.p. 230° C. (dec.). HPLC 84% [R_(t)=10.84 min., 20% MeOH, 80% water(with 0.1% DEA), 20 min.]; ¹H NMR (400 MHz, DMSO-d₆) δ 12.62 (s, 1H),11.47 (s, 1H), 10.21 (s, 1H), 8.72 (s, 1H), 8.04 (dd, J=7.9, 1.4 Hz,1H), 7.84 (d, J=8.8 Hz, 2H), 7.72 (d, J=8.7 Hz, 2H), 7.65-7.54 (m, 1H),7.23-7.20 (m, 1H); LC-MS (ESI−) m/z 417.02 (M−H−HCl)⁻; HRMS (ESI−) m/zcalculated for C₁₈H₁₃Cl₂N₄O₄ (M−H−HCl)⁻ 417.0163. found 417.0160.

N⁴-(2-Carboxyphenyl)-N-(4-carboxyphenyl)-5-chloropyrimidine-2,4-diaminehydrochloride (4d, method h in FIG. 4A)

A mixture of 3s (0.058 g, 0.119 mmol) in THF (0.4 mL) and NaOH (2 M aq.0.2 mL) was heated in a sealed tube at 110° C. (oil bath temperature)overnight. The THF was then removed under reduced pressure and HCl (1 Maq., 0.6 mL) was added to the residue. The resulting precipitate wasfiltered, washed with water (2 mL×3) and dried under vacuum. The solidobtained was then slurried in DMF (2 mL), filtered, washed with DMF (5mL), methanol (1 mL), Et₂O (3 mL) sequentially and dried under vacuum toafford the title compound 4d (0.036 g, 72%) as a white solid. m.p. 261°C. (dec.). ¹H NMR (400 MHz, DMSO-d₆) δ 12.59 (s, 1H), 11.45 (s, 1H),9.90 (s, 1H), 8.87 (s, 1H), 8.30 (s, 1H), 8.01-7.55 (m, 6H), 7.13 (s,1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.57 167.72, 157.79, 155.85, 155.36,145.16, 141.90, 134.51, 131.90, 130.84, 123.84, 122.82, 121.54, 118.76,117.26, 107.11; HPLC 97% [R_(t)=8.74 min., 15% MeOH, 85% water (with0.1% DEA) 20 min.]; LC-MS (ESI−) m/z 383.04 (M−H−HCl)⁻; HRMS (ESI−) m/zcalculated for C₁₈H₁₄ClN₄O₄ (M−H−HCl)⁻ 383.0553. found 383.0552.

N⁴-(2-Fluorophenyl)-N²-(4-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (6a, method m in FIG. 5A)

A mixture of 2-chloro-N-(2-fluorophenyl)pyrimidin-4-amine (5a) (0.096 g,0.428 mmol) and 4-aminobenzoic acid (0.069 g, 0.503 mmol) in EtOH (0.5mL) was heated in a microwave reactor at 150° C. for 20 min. The mixturewas filtered and the resulting precipitate was washed with EtOH (0.5mL×2) to provide the title compound 6a (0.107 g, 69%) as a white solid.m.p. 264° C. (dec). HPLC 98.7% (R_(t)=3.8 min., 55% MeOH in 0.1% TFAwater 20 min.); ¹H NMR (400 MHz, d₆-DMSO) δ 12.79 (s, 1H), 11.04 (s,1H), 10.87 (s, 1H), 8.13 (d, J=7.0 Hz, 1H), 7.77 (d, J=8.7 Hz, 2H), 7.66(t, J=7.9 Hz, 1H), 7.56 (d, J=8.5 Hz, 2H), 7.46-7.36 (m, 2H), 7.33-7.25(m, 1H), 6.58 (d, J=6.7 Hz, 1H); ¹⁹F NMR (400 MHz, DMSO-d₆) δ 121.12;¹³C NMR (100 MHz, DMSO-d₆) δ 167.40, 163.00, 156.59 (d, J=245 Hz),152.55, 145.08, 141.92, 130.79, 129.14, (d, J=9.27 Hz), 128.30, 126.43,125.39, 125.04 (d, J=12.15 Hz), 120.29, 116.88 (d, J=18.72 Hz), 99.84,LC-MS (ESI−) m/z 323.10 (M−H−HCl)⁻; HRMS (ESI−) m/z calculated forC₁₇H₁₂FN₄O₂ (M−H−HCl)⁻ 323.0950. found 323.0974.

N⁴-[2-(Trifluoromethyl)phenyl]-N²-phenylpyrimidine-2,4-diaminehydrochloride (6b, method m in FIG. 5A)

A mixture of chloropyrimidine 5f (0.076 g, 0.277 mmol) and aniline (0.03mL, 0.328 mmol) in EtOH (0.4 mL) was heated in a microwave reactor at150° C. for 20 min. The solvent was removed under reduced pressure toprovide an off-white solid. Ethyl acetate (3 mL) was added to thereaction mixture. The mixture was left at r.t. for 30 min and sonicatedoccasionally. The resulting precipitate was isolated by filtration andwashed with ethyl acetate (1 mL×5) and hexane (3 mL) to afford 6b (0.085g, 84%) as a white solid. m.p. 207° C. (dec). HPLC 100% (R_(t)=9.1 min.,55% MeOH in 0.1% TFA water 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 10.62(s, 1H), 10.43 (s, 1H), 8.01 (apparent s, 1H), 7.86-7.80 (m, 2H),7.71-7.48 (m, 2H), 7.27 (d, J=7.2 Hz, 2H), 7.14 (t, J=7.5 Hz, 2H), 7.03(t, J=7.3 Hz, 1H), 6.46 (s, 1H); LC-MS (ESI+) m/z 331.13 (M−Cl)⁺; HRMS(ESI+) m/z calculated for C₁₇H₁₄F₃N₄ (M−Cl)⁺ 331.1165. found 331.1170.

N⁴-(2-Chloro-4-fluorophenyl)-N²-(4-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (6c, method m in FIG. 5A)

A mixture of chloropyrimidine 5b (0.096 g, 0.372 mmol) and4-aminobenzoic acid (0.058 g, 0.422 mmol) in EtOH (0.5 mL) was heated ina microwave reactor at 150° C. for 20 min. The resulting precipitate wasisolated by filtration and washed with EtOH (0.5 mL×2), diethyl ether (2mL) and hexane (2 mL) sequentially to provide the title compound 6c(0.102 g, 69%) as a white solid. m.p. 268° C. (dec). HPLC 97.7%(R_(t)=4.8 min., 55% MeOH in 0.1% TFA water 20 min.); ¹H NMR (400 MHz,DMSO-d₆) δ 10.89 (s, 1H), 10.72 (s, 1H), 8.12 (d, J=7.0 Hz, 1H), 7.75(d, J=8.7 Hz, 2H), 7.72-7.63 (m, 2H), 7.50 (d, J=8.6 Hz, 2H), 7.40 (td,J=8.5, 2.9 Hz, 1H), 6.53 (d, J=7.0 Hz, 1H); ¹⁹F NMR (400 MHz, DMSO-d₆)112.66; ¹³C NMR (100 MHz, DMSO-d₆) δ 167.37 163.39, 161.22 (d, J=246.3Hz), 152.39, 145.37, 141.91, 131.87 (d, J=11.12 Hz); LC-MS (ESI−) m/z357.06 (M−H−HCl)⁻; HRMS (ESI−) m/z calculated for C₁₇H₁₁ClFN₄O₂(M−H−HCl)⁻ 357.0560. found 357.0521.

N⁴-[2-(Trifluoromethoxy)phenyl]-N²-(4-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (6d, method m in FIG. 5A)

A mixture of chloropyrimidine 5c (0.074 g, 0.255 mmol) and4-aminobenzoic acid (0.042 g, 0.306 mmol) in EtOH (0.5 mL) was heatedwith a microwave reactor at 150° C. for 20 min. The resultingprecipitate was isolated by filtration and washed with EtOH (0.5 mL×2),diethyl ether (2 mL) and hexane (2 mL) sequentially to provide the titlecompound 6d (0.63 g, 58%) as a white solid. m.p. 228° C. (dec). HPLC96.4% (R_(t)=5.8 min., 55% MeOH in 0.1% TFA water 20 min.); ¹H NMR (400MHz, DMSO-d₆) 10.93 (s, 1H), 10.75 (s, 1H), 8.12 (d, J=7.0 Hz, 1H),7.77-7.73 (m, 3H), 7.63-7.39 (m, 5H), 6.58 (d, J=6.7 Hz, 1H); ¹⁹F NMR(400 MHz, d₆-DMSO) δ 57.09; ¹³C NMR (100 MHz, DMSO-d₆) δ 167.40, 163.23,152.77, 145.96, 143.26, 142.04, 130.77, 130.16, 129.31, 129.10, 128.65,126.30, 122.51, 120.22, 118.10 (q, J=256 Hz), 99.76. LC-MS (ESI−) m/z389.07 (M−H−HCl)⁻; HRMS (ESI−) m/z calculated for C₁₈H₁₂F₃N₄O₃(M−H−HCl)⁻ 389.0867. found 389.0818.

N⁴-(2-Methoxyphenyl)-N²-(4-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (6e, method m in FIG. 5A)

A mixture of chloropyrimidine 5d (0.075 g, 0.317 mmol) and4-aminobenzoic acid (0.046 g, 0.328 mmol) in EtOH (0.5 mL) was heated ina microwave reactor at 150° C. for 20 min. Ethanol (0.5 mL) was added tothe reaction mixture. The resulting precipitate was isolated byfiltration and washed with EtOH (0.5 mL) and hexane (5 mL) to providethe title compound 6e (0.8 g, 68%) as a white solid. m.p. 247.5-249.3°C. HPLC 99.6% (R_(t)=6.0 min., 50% MeOH in 0.1% TFA water 20 min.); ¹HNMR (400 MHz, DMSO-d₆) δ 12.75 (s, 1H), 10.85 (s, 1H), 10.41 (s, 1H),8.03 (d, J=7.1 Hz, 1H), 7.77 (d, J=8.6 Hz, 2H), 7.59-7.56 (m, 3H), 7.33(t, J=7.9 Hz, 1H), 7.18 (d, J=8.3 Hz, 1H), 7.02 (t, J=7.5 Hz, 1H), 6.50(apparent s, 1H), 3.80 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.42,162.78, 153.64, 152.19, 144.09, 142.02, 130.81, 128.73, 127.42, 126.30,125.47, 120.89, 120.17, 112.66, 99.85, 56.32; LC-MS (ESI−) m/z 335.11(M−H−HCl)⁻; HRMS (ESI−) m/z calculated for C₁₈H₁₅N₄O₃ (M−H−HCl)⁻335.1150. found 335.1153.

N⁴-(2-Methoxyphenyl)-N²-(phenyl)pyrimidine-2,4-diamine (6f, method m inFIG. 5A)

A mixture of chloropyrimidine 5d (0.105 g, 0.444 mmol) and aniline (0.05mL, 0.548 mmol) in EtOH (0.5 mL) was heated in a microwave reactor at150° C. for 20 min. The solvent was removed under reduced pressure.Aqueous saturated NaHCO₃ (10 mL) was added to the residue and extractedwith ethyl acetate (10 mL×2). The combined organic phase was dried(Na₂SO₄), filtered and concentrated to dryness. The residue was purifiedby flash chromatography (10 g silica gel, Hex/EtOAc) to afford the titlecompound 6f (0.100 g, 76%) as an off-white solid. m.p. 142.3-144.5° C.HPLC 99.4% (R_(t)=4.90 min, 60% MeOH in 0.1% TFA water 20 min.); ¹H NMR(400 MHz, DMSO-d₆) δ 9.06 (s, 1H), 8.54 (s, 1H), 7.99 (d, J=8.0 Hz, 1H),7.96 (d, J=5.7 Hz, 1H), 7.69 (d, J=7.6 Hz, 2H), 7.18-7.14 (m, 2H),7.11-7.02 (m, 2H), 6.97-6.89 (m, 1H), 6.85 (t, J=7.3 Hz, 1H), 6.32 (d,J=5.8 Hz, 1H), 3.82 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) 161.00, 160.08,156.52, 149.55, 140.01, 128.97, 128.31, 123.69, 122.60, 121.50, 120.91,120.33, 110.62, 98.24; LC-MS (ESI+) m/z 293.15 (M+H)⁺; HRMS (ESI+) m/zcalculated for C₁₇H₁₇N₄O (M+H)⁺ 293.1397. found 293.1393.

N⁴-(2-Cyanophenyl)-N²-(phenyl)pyrimidine-2,4-diamine (6g, method m inFIG. 5A)

A mixture of chloropyrimidine 5e (0.092 g, 0.398 mmol) and aniline(0.037 ml, 0.398 mmol) in EtOH (0.5 mL) was heated in a microwavereactor at 150° C. for 20 min. The solvent was removed under reducedpressure. The solid obtained was slurried in ethyl acetate (3 mL),filtered, and washed with ethyl acetate (5 mL), and hexane (5 mL). Thedried solid was dissolved in methanol (3 mL). Diisopropylethylamine (1ml) was added and the solvent was removed under reduced pressure. Thesolid obtained was slurried with water (5 ml), filtered, washed withwater (10 ml×2), ether (5 ml), slurried with methanol (1 ml), filteredand dried under vacuum to provide the title compound 6g (0.027 g, 24%)as an off-white solid. m.p. 170.0.2-172.7° C. HPLC 100% [R_(t)=5.1 min.,50% MeOH, 50% water (with 0.1% TFA) 20 min.]; ¹H NMR (400 MHz, DMSO-d₆)δ 9.52 (s, 1H), 9.15 (s, 1H), 8.07 (d, J=5.7 Hz, 1H), 7.82 (d, J=8.8 Hz,1H), 7.77-7.66 (m, 2H), 7.59 (d, J=8.8 Hz, 2H), 7.32 (t, J=8.0 Hz, 1H),7.11 (t, J=7.9 Hz, 2H), 6.83 (t, J=7.3 Hz, 1H), 6.28 (d, J=5.7 Hz, 1H);LC-MS (ESI+) m/z 288.13 (M+H)⁺; HRMS (ESI+) m/z calculated for C₁₇H₁₄N₅(M+H)⁺ 288.1244. found 288.1241.

M-[2-(Trifluoromethyl)phenyl]-N²-(4-carboxyphenyl)pyrmidine-2,4-diaminehydrochloride (6h, method m in FIG. 5A): A mixture of chloropyrimidine5f (0.055 g, 0.2 mmol) and 4-aminobenzoic acid (0.03 g, 0.218 mmol) inEtOH (0.3 mL) was heated in a microwave reactor at 150° C. for 20 min.Ethanol (0.5 mL) was added to the reaction mixture and sonicated at roomtemperature for 5 min. The resulting precipitate was isolated byfiltration and washed with EtOH (0.5 mL×2), and hexane (3 mL×2) toafford the title compound 6h (0.067 g, 82%) as a white solid. m.p.240.0-242.6° C. HPLC 99.6% (R_(t)=7.7 min., 50% MeOH in 0.1% TFA water30 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 10.91 (s, 1H), 10.76 (s, 1H), 8.11(d, J=7.0 Hz, 1H), 7.90-7.85 (m, 2H), 7.69-7.64 (m, 4H), 7.38 (d, J=8.5Hz, 2H), 6.53 (d, J=6.7 Hz, 1H); ¹⁹F NMR (400 MHz, DMSO-d₆) δ 59.99 (s);¹³C NMR (100 MHz, d₆-DMSO) 167.35, 164.21, 152.47, 145.71, 142.05,135.37, 134.40, 131.77, 130.65, 129.09, 127. (q, J=4.6 Hz), 126.62 (q,J=29.5 Hz), 126.10, 124.03 (q, J=271.5 Hz), 119.77; LC-MS (ESI−) m/z373.08 (M+H)⁺; LC-MS (ESI+) m/z 375.11 (M+H)⁺; HRMS (ESI+) m/zcalculated for C₁₈H₁₄F₃N₄O₂ (M+H)⁺ 375.1063. found 375.1068.

N⁴-(2-Bromophenyl)-N-(4-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (61, method m in FIG. 5A)

A mixture of chloropyrimidine 5g (0.100 g, 0.350 mmol) and4-aminobenzoic acid (0.048 g, 0.350 mmol) in EtOH (0.5 mL) was heated ina microwave reactor at 150° C. for 20 min. The resulting precipitate wasisolated by filtration and washed with EtOH (0.5 mL×2) and dried undervacuum to provide the title compound 61 (0.069 g, 47%) as a white solid.m.p. 240° C. (dec.). HPLC 98% [R_(t)=7.40 min., 50% MeOH, 50% water(with 0.1% TFA) 20 min.]; ¹H NMR (400 MHz, DMSO-d₆) δ 10.46 (s, 2H),8.06 (d, J=6.8 Hz, 1H), 7.78 (d, J=8.3 Hz, 1H), 7.68 (d, J=8.5 Hz, 2H),7.59 (d, J=8.3 Hz, 1H), 7.51-7.47 (m, 3H), 7.32 (t, J=8.3 Hz, 1H), 6.45(s, 1H); ¹³C NMR (100 MHz, d₆-DMSO) 167.43, 163.07, 153.17, 146.60,142.37, 136.36, 133.80, 130.71, 129.95, 129.71, 129.19, 125.91, 121.27,119.83, 99.45; LC-MS (ESI+) m/z 385.02 (M−Cl)⁺; HRMS (ESI+) m/zcalculated for C₁₇H₁₄BrN₄O₂ (M−Cl)⁺ 385.0295. found 385.0292.

N⁴-(2-Chlorophenyl)-N²-(4-methylcarboxyphenyl)pyrimidine-2,4-diaminehydrochloride (6j, method I in FIG. 5A)

A mixture of chloropyrimidine 2l (0.120 g, 0.500 mmol) and4-aminophenylacetic acid (0.076 g, 0.5 mmol) in EtOH (2 mL with 1 dropof 1 M hydrochloric acid) was heated in a microwave reactor at 160° C.for 15 min. A clear solution was obtained. Ethanol was removed from themixture under vacuum, and the analysis of the crude NMR showed formationof 25% ethyl ester of 6j. The crude material was stirred in THF (0.5 mL)and NaOH solution (1 mL, 2 M) at r.t. for 16 h. The THF was evaporatedfrom the mixture and HCl (1 M) was added to acidify the mixture(pH=1-2). The precipitate obtained was filtered, washed with water (5mL) and dried under high vacuum to afford 6j (0.137 g, 70%) as a beigesolid. m.p. 132° C. (dec). HPLC 97% (R_(t)=3.77 min., 50% MeOH in 0.1%TFA water 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 12.19 (s, 1H disappearedon D₂O shake), 9.30 (s, 1H disappeared on D₂O shake), 9.21 (s, 1Hdisappeared on D₂O shake), 8.01 (d, J=6.0 Hz, 1H), 7.79 (d, J=8.0 Hz,1H), 7.54 (dd, J=8.0, 1.4 Hz, 1H partially overlapping), 7.51 (d, J=8.4Hz, 2H partially overlapping with dd), 7.38 (apptd, J=8.0, 1.4 Hz, 1H),7.24 (apptd, J=8.0, 1.4 Hz, 1H), 7.03 (d, J=8.4 Hz, 2H), 6.30 (d, J=6.0Hz, 1H), 3.45 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.65, 162.04,158.90, 155.15, 139.43, 136.41, 130.31, 129.87, 129.85, 128.56, 128.44,128.10, 126.90, 119.66, 119.63, 98.71; LC-MS (ESI+) m/z 355.11 (M−Cl)⁺;HRMS (ESI+) m/z calculated for C₁₈H₁₆ClN₄O₂ (M−Cl)⁺ 355.0956. found355.0971.

N⁴-(2-Chloromethyl)phenyl]-N²-(4-carboxy-3-hydroxyphenyl)pyrimidine-2,4-diaminehydrochloride (6k, methods n and o in FIG. 5A)

A mixture of chloropyrimidine 2l (0.100 g, 0.416 mmol) and methyl5-aminosalicylate (0.070 g, 0.416 mmol) in THF (10 mL) was stirred atroom temperature for 10 min. Two drops of concentrated HCl were addedand the mixture heated at reflux for 14 hours. The solvent was removedunder reduced pressure to provide a gray solid which was suspended insodium bicarbonate (sat. aq. 50 mL), sonicated for 10 min, filtered, andwashed with water (10 mL) to provide a white powder. The crude materialwas washed with methanol (2×10 ml) and finally with ethyl acetate (20ml) to give methyl4-(4-(2-chlorophenylamino)pyrimidin-2-ylamino)-2-hydroxybenzoate (6u)(0.081 g, 56%). ¹H NMR (400 MHz, d₆-DMSO) δ 10.61 (s, 1H), 9.63 (s, 1H),9.15 (s, 1H), 8.07 (s, 1H), 7.76 (s, 1H), 7.59-7.33 (3×broad s, 3H),7.25-7.10 (2×broad s, 2H), 6.35 (s, 1H), 3.83 (s, 3H). LC-MS (ESI+)371.09, m/z calculated for C₁₈H₁₆ClN₄O₃ (M+H)⁺ 371.0905. found 371.0914.To a stirred solution of ester 6u (200 mg, 0.540 mmol) in THF (15 ml),was added sodium hydroxide (108 mg, 2.70 mmol) in water (1.5 ml). Thereaction was heated under reflux for 14 hours. The solvent was removedunder reduced pressure to provide a white solid which was then dissolvedin water (20 ml) and acidified to pH ˜6-7 by addition of HCl (1 M). Thecolorless precipitate was filtered, washed with water and dried undervacuum. The crude solid was suspended in methanol (20 ml), sonicated for10 min., filtered, washed with methanol (5 ml) and dried under vacuum toprovide the title compound 6k (118 mg, 62%) as an off-white powder. m.p.202-204° C. HPLC 100% [R_(t)=8.3 min, 45% MeOH, 65% water (with 0.1%TFA) 20 min.]; ¹H NMR (400 MHz, DMSO-d₆) δ 13.55 (brs, 1H), 11.29 (brs,1H), 10.14 (brs, 1H), 9.93 (s, 1H), 8.07 (d, J=6.7 Hz, 1H), 7.70 (d,J=7.8 Hz, 1H), 7.57 (d, J=7.6 Hz, 1H), 7.53 (d, J=8.5 Hz, 1H), 7.41 (t,J=7.8 Hz, 1H), 7.30 (t, J=8.0 Hz, 1H), 7.25 (s, 1H), 7.02 (d, J=8.5 Hz,1H), 6.43 (d, J=5.7 Hz, 1H); ¹³C NMR (100 MHz, d₆-DMSO), δ 172.34,162.82, 162.58, 155.69, 146.06, 135.35, 131.18, 130.58, 129.32, 128.64,128.53, 128.25, 111.11, 107.27, 106.54, 99.78; LC-MS (ESI+) m/z 357.08(M−Cl)⁺; HRMS (ESI+) m/z calculated for C₁₇H₁₄ClN₄O₃ (M−Cl)⁺ 357.0749.found 357.0751.

N⁴-(2-Fluorophenyl)-N²-4-phenylpyrimidine-2,4-diamine (61, method m inFIG. 5A)

A mixture of chloropyrimidine 5a (0.073 g, 0.325 mmol) and aniline (0.04mL, 0.438 mmol) in EtOH (0.5 mL) was heated in a microwave reactor at150° C. for 20 min. The solvent was removed under reduced pressure.Aqueous saturated NaHCO₃ (10 mL) was added to the residue and extractedwith ethyl acetate (10 mL×2). The combined organic phase was dried(Na₂SO₄), filtered and concentrated to dryness. The residue was purifiedby flash chromatography (10 g silica gel, hex/EtOAc) to afford the titlecompound 6l (0.050 g, 55%) as an off-white solid. m.p. 133.2-134.6° C.HPLC 99.5% (R_(t)=4.4 min, 60% MeOH in 0.1% TFA water 20 min.); ¹H NMR(400 MHz, DMSO-d₆) δ 9.11 (s, 1H), 9.07 (s, 1H), 8.02 (d, J=5.7 Hz, 1H),7.97 (t, J=7.9 Hz, 1H), 7.65 (d, J=8.6 Hz, 2H), 7.32-7.23 (m, 1H),7.22-7.07 (m, 4H), 6.86 (t, J=7.3 Hz, 1H), 6.30 (d, J=5.7 Hz, 1H); LC-MS(ESI+) m/z 281.11 (M−Cl)⁺; HRMS (ESI+) m/z calculated for C₁₆H₁₄FN₄(M−Cl)⁺ 281.1197. found 281.1209.

N⁴-(2-Iodophenyl)-N²-(4-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (6m, method m in FIG. 5A)

A mixture of chloropyrimidine 5h (0.119 g, 0.358 mmol) and4-aminobenzoic acid (0.048 g, 0.350 mmol) in EtOH (0.5 mL) was heated ina microwave reactor at 150° C. for 20 min. The solvent was then removedunder reduced pressure to provide an off-white solid. The solid wasslurried in methanol (2 mL), filtered, washed with methanol (2 mL), anddried under vacuum to provide the title compound 6m (0.053 g, 32%) as awhite solid. m.p. 253° C. (dec). HPLC 99.7% [R_(t)=7.80 min., 50% MeOH,50% water (with 0.1% TFA) 20 min.]; ¹H NMR (400 MHz, DMSO-d₆) δ 12.76(bs, 1H), 10.72 (s, 1H), 10.62 (s, 1H), 8.07 (d, J=7.0 Hz, 1H), 8.01(dd, J=1.2, 7.9 Hz, 1H), 7.67 (d, J=8.5 Hz, 2H), 7.54-7.45 (m, 4H),7.19-7.15 (m, 1H), 6.44 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.36,163.09, 152.32, 145.44, 142.04, 139.99, 139.67, 130.68, 130.04, 129.94,129.45, 126.12, 119.90, 99.24; LC-MS (ESI+) m/z 433.02 (M−Cl)⁺; HRMS(ESI+) m/z calculated for C₁₇H₁₄N₄O₂ (M−Cl)⁺ 433.0156. found 433.0150.

N⁴-(2-Cyanophenyl)-N-(4-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (6n, method m in FIG. 5A)

A mixture of chloropyrimidine 5e (0.094 g, 0.406 mmol) and4-aminobenzoic acid (0.037 mg, 0.408 mmol) in EtOH (0.5 mL) was heatedwith in a microwave reactor at 150° C. for 20 min. The solvent wasremoved under reduced pressure. The compound was purified by reversephase C-18 preparative HPLC [Eclipse XDB-C18 PrepHT 21.2×250 mm, 7 μm;40% MeOH, 60% water (with 0.1% TFA) 20 min, 20 mL/min.] to provide thetitle compound as its TFA salt. The solid obtained was suspended inmethanol (9 mL) followed by the addition of HCl (4 M in dioxane, 1 mL).The solvent was then removed in a Genevac evaporator. The dried solidwas slurried with acetonitrile (1 mL), filtered, washed withacetonitrile (1 mL) and dried under vacuum to provide the title compound6n (0.022 g, 15%) as an off-white solid. m.p. 222° C. (dec). HPLC 97.6%[R_(t)=5.5 min., 40% MeOH, 60% water (with 0.1% TFA) 20 min.]; ¹H NMR(400 MHz, DMSO-d₆) δ 10.67 (s, 1H), 10.43 (s, 1H), 8.14 (d, J=6.8 Hz,1H,), 7.93 (d, J=7.6 Hz, 1H), 7.80 (t, J=7.6 Hz, 1H), 7.72-7.68 (m, 3H),7.55 (d, J=7.3 Hz, 2H), 7.50 (t, J=7.6 Hz, 1H), 6.50 (d, J=6.8 Hz, 1H),LC-MS (ESI+) m/z 332.11 (M−Cl)⁺; HRMS (ESI+) m/z calculated forC₁₈H₁₄N₅O₂ (M−Cl)⁺ 332.1142. found 332.1139.

N⁴-(2-Chlorophenyl)-N²-(3-carboxyphenyl)pyrimidine-2,4-diaminehydrochloride (6o, method m in FIG. 5A)

A mixture of 21 (0.100 g, 0.417 mmol) and 3-aminobenzoic acid (0.057 g,0.417 mmol) in EtOH (0.5 mL) was heated in a microwave reactor at 150°C. for 20 min. The precipitate obtained upon cooling the reactionmixture was filtered and washed quickly with ethanol (3 ml) to obtainthe 6o (0.097 g, 62%) as a white solid. m.p. 290-296° C. (dec). HPLC100% (R_(t)=7.58 min., 50% MeOH in 0.1% TFA water, 20 min.); ¹H NMR (400MHz, DMSO-d₆) δ 13.03 (s, 1H), 10.49 (brs, 1H), 8.04 (d, J=6.4 Hz, 1H),7.88 (brs, 1H), 7.73 (appd, J=7.6 Hz, 1H), 7.65 (d, J=8.0 Hz, 1H), 7.60(d, J=7.6 Hz, 1H), 7.57 (dd, J=7.9, 1.5 Hz, 1H), 7.37 (td, J=7.6, 0.8Hz, 1H), 7.32-7.27 (m, 2H), 6.49 (s, 1H); ¹³C NMR (101 MHz, DMSO) δ167.5, 162.95, 153.3, 145.9, 138.08, 134.6, 132.1, 130.5, 129.6, 129.5,129.1, 128.89, 128.36, 125.9, 125.5, 122.6, 99.5; elemental analysiscalculated for C₁₇H₁₄Cl₂N₄O₂: C, 54.13; H, 3.74; N, 14.85. Found: C,53.78; H, 3.64; N, 14.66.

N⁴-(2-Chlorophenyl)-N²-(4-carbamoyl)pyrimidine-2,4-diamine hydrochloride(6p, method l in FIG. 5A)

A mixture of chloropyrimidine 21 (0.100 g, 0.417 mmol) and4-aminobenzamide (0.057 g, 0.417 mmol) in EtOH (2.0 mL with 1 drop of 1M hydrochloric acid) was heated with a microwave reactor at 160° C. for15 min. The mixture was filtered and the solid obtained was washed withMeOH (1 mL) and dried to afford the desired compound 6p (0.110 g, 70%)as a white solid. m.p. 227° C. (dec.). HPLC 99% (R_(t)=3.70 min., 50%MeOH in 0.1% TFA water 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 10.75 (brs,2H disappeared on D₂O shake), 8.09 (dd, J=6.1, 3.5 Hz, 1H), 7.91 (brs,1H disappeared on D₂O shake), 7.71-7.64 (m, 4H), 7.50-7.42 (m, 4H), 7.30(brs, 1H disappeared on D₂O shake), 6.52 (brs, 1H); ¹³C NMR (100 MHz,DMSO-d₆) δ 167.76, 163.15, 152.33, 144.97, 140.39, 134.55, 130.62,130.29, 130.02, 129.54, 129.49, 128.90, 128.55, 119.98, 99.43; LC-MS(ESI+) m/z 340.10 (M−Cl)⁺; HRMS (ESI+) m/z calculated for C₁₇H₁₅ClN₅O(M−Cl)⁺ 340.0960. found 340.0971.

N⁴-(2-Biphenyl)-N²-(4-carboxyphenyl)pyrimidine-2,4-diamine hydrochloride(6q, method m in FIG. 5A)

A mixture of chloropyrimidine 5i (0.096 g, 0.34 mmol) and 4-aminobenzoicacid (0.054 g, 0.393 mmol) in EtOH (0.4 mL) was heated in a microwavereactor at 150° C. for 20 min. The resulting precipitate was filteredand washed with EtOH (0.5 mL×3) to provide the title compound 6q (0.098g, 67%), as a white solid. m.p. 259° C. (dec). HPLC 100% (R_(t)=8.7min., 55% MeOH in 0.1% TFA water 20 min.); ¹H NMR (400 MHz, DMSO-d₆)12.83 (s, 1H), 10.96 (s, 1H), 10.71 (s, 1H), 7.96 (d, J=7.2 Hz, 1H),7.73 (d, J=8.3 Hz, 2H), 7.61-7.42 (m, 6H), 7.42-7.17 (m, 5H), 6.31(apparent s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.41, 163.26, 152.00,144.30, 142.07, 138.96, 134.37, 131.35, 130.75, 129.35, 129.09, 128.74,128.17, 126.18, 119.89, 99.22; LC-MS (ESI−) m/z 381.13 (M−H−HCl)⁻; HRMS(ESI−) m/z calculated for C₂₃H₁₇N₄O₂ (M−H−HCl)⁻ 381.1357. found381.11366.

N⁴-(2-Chlorophenyl)-N²-(4-carboxyphenyl)-N⁴-methylpyrimidine-2,4-diaminehydrochloride (6r, method m in FIG. 5A)

This was obtained as a white solid (0.127 g, 0.36 mmol, 80%) from 5j(0.115 g, 0.452 mmol) and 4-aminobenzoic acid (0.062 g, 0.452 mmol) inthe same manner as described for 6s. m.p. 288° C. (dec.). HPLC 98%[R_(t)=8.39 min., 50% MeOH, 50% water (with 0.1% TFA) 20 min.]. ¹H NMR(400 MHz, CD₃OD) δ 8.17-8.04 (m, 2.57H), 7.77-7.74 (m, 3H), 7.70-7.67(m, 2H), 7.58-7.54 (m, 4H), 7.49-7.41 (m, 0.42H), 7.17 (d, J=8.8 Hz, 1H,major), 6.83 (d, J=7.5 Hz, 0.41H, minor), 5.80 (d, J=7.4 Hz, 1H, major),3.55 (s, 4H); LC-MS (ESI+) m/z 355.10 (M−Cl)⁺; HRMS (ESI+) m/zcalculated for C₁₈H₁₆ClN₄O₂ (M−Cl)⁺ 355.0956. found 355.0962; ElementalAnalysis: Found C, 54.98%; H, 4.12; N, 14.18. C₁₈H₁₆C₁₂N₄O₂ requires C,55.26%; H, 4.12; N, 14.32.

N⁴-(2-Chlorophenyl)-(4-carboxyphenyl)-ethyl-ethylpyrimidine-2,4-diaminehydrochloride (6s, method m in FIG. 5A)

A solution of 5k (0.151 g, 0.673 mmol) and 4-aminobenzoic acid (0.092 g,0.674 mmol) in anhydrous ethanol (0.673 mL) was heated in a microwavereactor at 150° C. for 20 min. The resulting precipitate was isolated byfiltration and washed with EtOH (1 mL×2) and dried under vacuum toprovide the title compound 6s (0.172 g, 71%), as a white solid. m.p.284° C. (dec). HPLC 100% [R_(t)=13.96 min., 50% MeOH, 50% water (with0.1% TFA) 20 min.]. Two rotamers are present: ¹H NMR (400 MHz, CD₃OD) δ8.11 (d, J=8.8 Hz, 2H, major), 8.07 (d, J=7.6 Hz, 0.4H, minor),7.75-7.66 (m, 5H), 7.63-7.54 (m, 5H), 7.15 (d, J=8.8 Hz, 1H, major),6.87 (d, J=7.5 Hz, 0.5H, minor), 5.74 (d, J=7.4 Hz, 1H, major),4.30-4.21 (m, 1H), 4.09-4.02 (m, 0.6H, minor), 3.93-3.83 (m, 2H), 1.35(t, J=7.2 Hz, 1.5H, minor), 1.29 (t, J=7.2 Hz, 3H, major); LC-MS (ESI+)m/z 369.12 (M−Cl)⁺; HRMS (ESI+) m/z calculated for C₁₉H₁₈ClN₄O₂ (M−Cl)⁺369.1113. found 369.1123; Elemental Analysis: Found C, 56.30%; H, 4.45;N, 13.76. C₁₉H₁₈Cl₂N₄O₂ requires C, 56.31%; H, 4.48; N, 13.82.

N⁴-(2-Chlorophenyl)-N²-(3-carboxyphenyl)-5-fluoropyrimidine-2,4-diaminehydrochloride (6t, method m in FIG. 5A)

A mixture of 2k (0.100 g, 0.350 mmol) and 3-aminobenzoic acid (0.048 g,0.350 mmol) in EtOH (0.5 mL) was heated in a microwave reactor at 150°C. for 20 min. The reaction mixture was cooled and the precipitateobtained was filtered and washed quickly with ethanol (3 ml) to provide6t (0.080 g, 57%) as a white solid. m.p. 257° C. (dec.). HPLC 98%(R_(t)=9.45 min., 50% MeOH in 0.1% TFA water, 20 min.); ¹H NMR (400 MHz,DMSO-d₆) δ 12.74 (brs, 1H), 9.32 (s, 1H), 9.18 (s, 1H), 8.12 (d, J=3.5Hz, 1H), 8.02 (s, 1H), 7.80 (d, J=8.2 Hz, 1H), 7.61 (d, J=7.8 Hz, 1H),7.56 (d, J=7.9 Hz, 1H), 7.40-7.36 (m, 2H), 7.29 (t, J=7.6 Hz, 1H), 7.11(t, J=7.9 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.14, 156.0 (d, J=2.9Hz), 151.37 (d, J=11.5 Hz), 141.75, 141.43 (d, J=246.44 Hz), 141.39 (d,J=18.4 Hz), 135.92, 131.54, 130.88, 130.38, 129.55, 128.90, 128.32,128.03, 122.84, 122.18, 119.87; ¹⁹F NMR (376 MHz, DMSO) δ −165.76; LC-MS(ESI−) m/z 357.06 (M−H−HCl)⁻; HRMS (ESI−) m/z calculated forC₁₇H₁₂ClFN₄O₂ (M−H−HCl)⁻ 357.0560. found 357.0554.

N⁴-(2-Chlorophenyl)-N²-[4-N-(2-morpholinoethyl)carbamoylphenyl]pyrimidine-2,4-diaminedihydro-chloride (9a, method r in FIG. 7A)

A solution of 2l (0.100 g, 0.42 mmol) and 8a (0.105 g, 0.42 mmol) inisopropyl alcohol (8 mL) and concentrated HCl (0.315 mL) was heated in amicrowave reactor at 170° C. for 20 min. The solvent was then removedunder reduced pressure. The resulting solid was dissolved in methanol (2mL) followed by the addition of diethyl ether (10 mL). The precipitatewas filtered and dried under vacuum to afford the title compound 9a(0.054 g, 29%) as white crystals. m.p. 173.0-174.6° C. HPLC 99.0%[R_(t)=5.53 min., 50% MeOH, 40% water (with 0.1% TFA) 20 min.]; ¹H NMR(400 MHz, DMSO-d₆) δ 11.37-10.62 (m, 1H), 8.88 (s, 1H), 8.59-8.43 (m,1H), 8.12 (s, 1H), 7.78 (s, 2H), 7.69-7.61 (m, 2H), 7.50 (s, 2H), 7.43(s, 1H), 6.76-6.41 (m, 2H), 4.03-2.95 (m, 12H); ¹³C NMR (100 MHz,DMSO-d₆) δ 166.39, 163.08, 152.86, 145.97, 140.91, 134.67, 130.67,130.15, 129.42, 129.24, 128.82, 128.60, 119.81, 99.46, 63.80, 56.32,51.87, 34.28. LC-MS (ESI+) m/z 453.17 (M−HCl-Cl)⁺; HRMS (ESI+) m/zcalculated for C₂₃H₂₅ClN₆O₂ (M−HCl-Cl)⁺ 453.1800. found 453.1797.

N⁴-(2-Chlorophenyl)-N²-[4-N-(2-dimethylaminoethyl)carbamoylphenyl]-pyrimidine-2,4-diaminedihydro-chloride (9b, method r in FIG. 7A)

This was obtained as a white solid (0.02 g, 0.04 mmol, 8%) from 21(0.100 g, 0.48 mmol) and 8b (0.116 g, 0.48 mmol) in a similar manner asdescribed for 9a. m.p. 143.1-145.8° C. HPLC 98% [R_(t)=5.10 min., 60%MeOH, 40% water (with 0.1% TFA) 20 min.]; ¹H NMR (400 MHz, DMSO-d₆) δ10.65 (s, 1H), 9.96 (s, 1H), 8.75 (s, 1H), 8.09 (s, 1H), 7.73 (s, 2H),7.71-7.61 (m, 2H), 7.58-7.45 (m, 3H), 7.45-7.35 (m, 1H), 6.50 (s, 1H),3.62-3.55 (m, 2H), 3.23 (d, J=5.7 Hz, 2H), 2.80 (d, J=4.0 Hz, 6H); ¹³CNMR (100 MHz, DMSO-d₆) δ 166.50, 163.10, 152.72, 145.80, 140.87, 134.73,130.67, 130.19, 129.45, 129.31, 128.61, 119.80, 99.50, 56.76, 43.01,35.16; LC-MS (ESI+) m/z 411.18 (M−HCl-Cl)⁺; HRMS (ESI+) m/z calculatedfor C₂₁H₂₄ClN₆O (M−HCl-Cl)⁺ 411.1695. found 411.1693.

N⁴-(2-Chlorophenyl)-N²-[4-N-(2-methoxyethyl)carbamoylphenyl]pyrimidine-2,4-diamine hydrochloride (9c, method s in FIG. 7A)

This was obtained as a white solid (0.047 g, 0.12 mmol) from 21 (0.092g, 0.47 mmol) and 8c (0.060 g, 0.47 mmol) in a similar manner asdescribed for compound 6s m.p. 210.4-213.0° C. HPLC 97% [R_(t)=5.61min., 50% MeOH, 50% water (with 0.1% TFA) 20 min.]; ¹H NMR (400 MHz,DMSO-d₆) δ 10.71 (s, 2H), 8.44 (t, J=5.6 Hz, 1H), 8.08 (d, J=7.3 Hz,1H), 7.68-7.62 (m, 4H), 7.49-7.38 (m, 3H), 6.52 (s, 1H), 3.41-3.37 (m,4H), 3.24 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 166.06, 163.12, 152.41,145.17, 140.32, 134.55, 130.60, 130.23, 130.08, 129.50, 129.42, 128.56,119.98, 99.48, 71.50, 58.58, 58.55 LC-MS (ESI+) m/z 398.14 (M−Cl)⁺. HRMS(ESI+) m/z calculated for C₂₀H₂₁ClN₅O₂ (M−Cl)⁺ 398.1378. found 398.1370.

N⁴-(2-Chlorophenyl)-N²-[4-(methylsulfonyl)phenyl]pyrimidine-2,4-diamine(9d, method t in FIG. 7A)

A solution of 2l (0.098 g, 0.41 mmol) and 4-(methylsulfonyl)aniline(0.070 g, 0.41 mmol) in ethanol (1 mL) and 1M HCl (aq., 1.0 mL) washeated in a microwave reactor at 180° C. for 15 min. The solvent wasremoved under reduced pressure. The solid obtained was slurred in asaturated solution of sodium bicarbonate filtered, slurried withmethanol (1 mL), then filtered to yield the title compound 9d (0.077 g,44%) as a white solid. m.p. 169.3-171.3° C. HPLC 99% [R_(t)=4.15 min.,50% MeOH, 50% water (with 0.1% TFA) 20 min.]; ¹H NMR (400 MHz, DMSO-d₆)δ 9.72 (s, 1H), 9.17 (s, 1H), 8.08 (d, J=5.7 Hz, 1H), 7.84 (d, J=8.7 Hz,2H), 7.74 (d, J=7.8, 1H), 7.65-7.51 (m, 3H), 7.41 (t, J=7.6, 1H), 7.26(t, J=7.6 Hz, 1H), 6.35 (d, J=5.7 Hz, 1H), 3.09 (s, 3H); ¹³C NMR (100MHz, DMSO-d₆) δ 162.03, 159.47, 156.92, 146.25, 136.52, 132.20, 130.40,129.03, 128.38, 128.34, 128.21, 127.12, 118.38, 99.77, 44.70; LC-MS(ESI+) m/z 375.06 (M+H)⁺; HRMS (ESI+) m/z calculated for C₁₇H₁₆ClN₄O₂S(M+H)⁺ 375.0677. found 375.0670.

N⁴-(2-Chlorophenyl)-N²-(3-acetamidophenyl)pyrimidine-2,4-diaminehydrochloride (9e, method s in FIG. 7A)

This compound was prepared from 21 (0.105 g, 0.44 mmol) andN-(3-aminophenyl)acetamide (0.066 g, 0.44 mmol) in a similar manner asdescribed for 6s. The solvent was then removed under reduced pressure.The resulting solid was dissolved in methanol (2 ml), followed by theaddition of ethyl acetate (10 ml). A precipitate formed and the mixturewas sonicated for 30 min at room temperature. The precipitate wasfiltered and washed with ethyl acetate (10 mL) and hexane (20 mL),affording the pure product 9e (0.166 g, 95%), as an off-white solid.m.p. 150° C. (dec). HPLC 100% [R_(t)=5.30 min., 50% MeOH, 50% water(with 0.1% TFA) 20 min.]; ¹H NMR (400 MHz, DMSO-d₆) δ 10.63 (s, 1H),10.45 (s, 1H), 10.00 (s, 1H), 8.00 (d, J=5.0 Hz, 1H), 7.70-7.52 (m, 3H),7.4-7.28 (m, 2H), 7.24-7.13 (m, 2H), 7.13-7.07 (m, 1H), 2.02 (s, 3H);LC-MS (ESI+) m/z 354.11 (M−Cl)⁺; HRMS (ESI+) m/z calculated forC₁₈H₁₇ClN₅O (M−Cl)⁺ 354.1116. found 354.1119.

N⁴-(2-Chlorophenyl)-N²-[6-(1H-benzo[d]imidazol-2(3H)-one)]pyrimidine-2,4-diaminehydrochloride (9f, method s in FIG. 7A)

A solution of 2l (0.105g, 0.437 mmol) and5-amino-1H-benzo[d]imidazol-2(3H)-one (0.065 g, 0.437 mmol) and ethanol(0.437 mL) was heated in a microwave reactor at 150° C. for 40 min. Theresulting precipitate was filtered, dried under vacuum and suspended inethanol. The suspension was sonicated for thirty minutes, filtered anddried under vacuum to provide the title compound 9f (0.112 g, 66%) as anoff-white solid. m.p. 263° C. (dec). HPLC 98% [R_(t)=3.21 min., 50%MeOH, 50% water (with 0.1% TFA) 20 min.]; ¹H NMR (400 MHz, CD₃OD) δ 7.74(s, 2H), 7.65-7.63 (m, 1H), 7.54-7.52 (m, 1H), 7.35-7.28 (m, 2H), 7.04(d, J=7.4 Hz, 3H), 6.42 (s, 1H); LC-MS (ESI+) m/z 353.10 (M−Cl)⁺; HRMS(ESI+) m/z calculated for C₁₇H₁₄ClN₆O (M−Cl)⁺ 353.0912. found 353.0913.

N⁴-(2-Chlorophenyl)-N²-(4-carboxyl-3-methoxyphenyl)pyrimidine-2,4-diaminehydrochloride (9g, method s in FIG. 7A)

A mixture of chloropyrimidine 2l (0.100 g, 0.416 mmol) and4-amino-2-methoxybenzoic acid (0.070 g, 0.418 mmol) in EtOH (0.5 mL) washeated with a microwave reactor at 150° C. for 20 min. The solvent wasevaporated from the resulting thick mass under reduced pressure. Theresidue was suspended in ethyl acetate (8 mL) and sonicated for 10 min.The mixture was filtered and the solid was washed with ethyl acetate (8mL) and ethyl acetate:methanol (1:1, 1 mL) to provide the title compound9g (0.068 g, 57%) as a gray solid. m.p. 160-162° C. HPLC 95% [R_(t)=4.5min, 50% MeOH, 50% water (with 0.1% TFA) 20 min.]; ¹H NMR (400 MHz,DMSO-d₆), δ 12.69 (s, 1H), 10.42 (s, 1H), 10.24 (s, 1H), 7.95 (d, J=6.2Hz, 1H), 7.64-7.51 (m, 3H), 7.48 (d, J=8.4 Hz, 1H), 7.36 (t, J=7.7 Hz,1H), 7.29 (t, J=7.4 Hz, 1H), 6.98 (d, J=8.8 Hz, 1H), 6.45 (s, 1H), 3.76(s, 3H); ¹³C NMR (100 MHz, DMSO-d₆), δ 167.37, 162.97, 155.67, 152.95,144.61, 134.39, 130.51, 129.61, 129.00, 128.35, 127.11, 124.90, 122.22,113.36, 99.26, 56.70; LC-MS (ESI+) m/z 371.09 (M−Cl)⁺; HRMS (ESI+) m/zcalculated for C₁₈H₁₆ClN₄O₃ (M−Cl)⁺ 371.0905. found 371.0909.

N⁴-(2-Chlorophenyl)-N²-[4-(N-morpholino)phenyl]pyrimidine-2,4-diaminehydrochloride (9h, method t in FIG. 7A)

A mixture of chloropyrimidine 2l (0.120 g, 0.500 mmol) and4-morpholinoaniline (0.089 g, 0.500 mmol) in EtOH (2 mL with 1 drop of 1M of hydrochloric acid) was heated in a microwave reactor at 160° C. for15 min. The reaction mixture was cooled to r.t. and the productprecipitated. The mixture was filtered, and the product obtained wasquickly washed with MeOH (1 mL), DCM (1 mL) and dried to afford 9h (0.14g, 67%) as a green solid. m.p. 136-138° C. HPLC 99% (R_(t)=7.43 min.,50% MeOH in 0.1% TFA water 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 12.11(s, 1H disappeared on D₂O shake), 10.63 (s, 1H disappeared on D₂Oshake), 10.37 (s, 1H disappeared on D₂O shake), 7.94 (brs, 1H),7.65-0.60 (m, 2H), 7.43 (appt, J=7.6 Hz, 1H), 7.36 (appt, J=7.6 Hz, 1H),7.22 (d, J=8.1 Hz, 2H), 6.84 (brs, 2H), 6.45 (brs, 1H), 3.73 (appt,J=4.4 Hz, 4H), 3.05 (brt, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.03,152.56, 148.63, 144.36, 134.58, 130.53, 129.99, 129.37, 129.12, 128.43,122.90, 116.05, 98.78, 66.65, 49.38; LC-MS (ESI+) m/z 382.15 (M−Cl)⁺;HRMS (ESI+) m/z calculated for C₂₀H₂₁ClN₅O (M−Cl)⁺ 382.1429. found382.1434.

N⁴-(2-Chlorophenyl)-N²-[4-(aminosulfonyl)phenyl]pyrimidine-2,4-diaminehydrochloride (9i, method t in FIG. 7A)

A mixture of chloropyrimidine 21 (0.120 g, 0.500 mmol) and4-aminobenzenesulfonamide (0.086 g, 0.500 mmol) in EtOH (2 mL with 1drop of 1 M HCl) was heated in a microwave reactor at 160° C. for 15min. The precipitate formed upon cooling filtered and washed with MeOH(2 mL), then slurried and sonicated in DCM (5 mL) and filtered. Theproduct obtained was dried to afford 91 (0.155 g, 75%) as a white solid.m.p. 215° C. (dec.). HPLC 96% (R_(t)=3.23 min., 50% MeOH in 0.1% TFAwater 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 10.81 (s, 1H), 10.67 (s,1H), 8.10 (d, J=6.9 Hz, 1H), 7.67-7.63 (m, 2H), 7.61-7.56 (m, 4H), 7.47(td, J=7.7, 1.6 Hz, 1H), 7.41 (td, J=7.7, 1.6 Hz, 1H), 7.28 (s, 2H),6.53 (appd, J=5.2 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.15, 152.43,145.28, 140.86, 139.52, 134.61, 130.64, 130.38, 129.60, 129.50, 128.57,127.10, 120.32, 99.66; LC-MS (ESI+) m/z 376.06 (M−Cl)⁺; HRMS (ESI+) m/zcalculated for C₁₆H₁₅ClN₅O₂S (M−Cl)⁺ 376.0629. found 376.0634.

N⁴-(2-Chlorophenyl)-N²-[4-(N-morpholino)phenyl]-5-fluoropyrimidine-2,4-diamine(9j, method s in FIG. 7A)

A mixture of chloropyrimidine 2k (0.100 g, 0.387 mmol) and4-morpholinoaniline (0.069 g, 0.387 mmol) in ethanol (1 mL) was heatedin a microwave reactor at 150° C. for 20 min. The reaction mixture wascooled, and stirred at room temperature for 30 min. The solvent wasremoved in vacuo. The residual solid was suspended in ethyl acetate (10mL) and sonicated for 10 min, filtered and washed with ethyl acetate (5mL). The solid was suspended in sodium bicarbonate (sat. aq. 50 mL),filtered, and washed with water (10 mL) to provide the title compound 9k(0.92 g, 60%) as an off-white solid. m.p. 209-211° C. HPLC 99%[R_(t)=7.2 min., 50% MeOH, 50% water (with 0.1% TFA) 20 min.]; ¹H NMR(400 MHz, DMSO-d₆) δ 9.63 (s, 1H), 9.36 (s, 1H), 8.11 (d, J_(H-F)=4.2Hz, 1H), 7.61-7.54 (m, 2H), 7.44-7.37 (m, 1H), 7.36-7.25 (m, 3H), 6.79(s, 2H), 3.73 (s, 4H), 3.03 (s, 4H). ¹H NMR (400 MHz, DMSO-d₆+D₂O) δ8.06 (d, J=4.1 Hz, 1H), 7.59-7.51 (m, 2H), 7.39 (td, J=7.6, 1.5 Hz, 1H),7.32 (td, J=7.7, 1.7 Hz, 1H), 7.27 (d, J=9.0 Hz, 2H), 6.79 (d, J=8.9 Hz,2H), 3.71 (t, J=4.0 Hz, 4H), 3.03 (t, J=4.4 Hz, 4H); ¹⁹F NMR (376 MHz,DMSO-d₆), δ −165.59; ¹³C NMR (100 MHz, DMSO-d₆) δ 154.38, 152.31 (d,J=12.0 Hz), 141.63, 139.04, 139.19, 135.42, 134.20, 130.43, 129.76,120.74, 117.11, 66.31, 50.71; LC-MS (ESI+) m/z 400.13 (M−Cl)⁺; HRMS(ESI+) m/z calculated for C₂₀H₂₀ClFN₅O (M−Cl)⁺ 400.1335. found 400.1344.

4-(2-Chlorophenylamino)-2-(4-(morpholinomethyl)phenyl)-5-fluoropyrimidine(9k, method u in FIG. 7A)

A mixture of chloropyrimidine 2k (0.100 g, 0.387 mmol),4-(morpholinomethyl)aniline (0.087 g, 0.452 mmol), X-Phos(2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl) (0.019 g, 0.0387mmol), bis(dibenzylideneacetone) palladium(0) (0.022 g, 0.0387 mmol),potassium carbonate (0.117 g, 0.851 mmol) in tert-BuOH (3.0 mL) washeated under reflux for 18 hours. The solvent was evaporated from theresulting dark solution. The dark residue was purified by chromatography(SiO₂, ethyl acetate and hexanes) to provide the title compound 9k(0.062 g, 39%) as an off-white solid. m.p. 142-144° C. HPLC 98%[R_(t)=23.4 min., 50% MeOH, 50% water (with 0.1% TFA) 36 min.]; ¹H NMR(400 MHz, DMSO-d₆), δ 9.16 (s, 1H), 9.10 (s, 1H), 8.07 (d, J_(H-F)=2.6Hz, 1H), 7.58 (t, J=6.8 Hz, 2H), 7.42-7.34 (m, 3H), 7.31 (t, J=8.2 Hz,1H), 6.94 (d, J=7.7 Hz, 2H), 3.52 (s, 4H), 2.26 (s, 4H); ¹³C NMR (100MHz, DMSO-d₆), δ 156.22 (d, J_(CF)=2.8 Hz), 151.27 (d, J_(CF)=11.4 Hz),141.15 (d, J_(CF)=245 Hz), 141.62 (d, J_(CF)=18.9 Hz), 140.44, 136.11,131.08, 130.33, 130.26, 129.73, 129.52, 128.26, 128.01, 118.57, 66.84,62.79, 53.74; ¹⁹F NMR (376 MHz, DMSO-d₆), δ −166.63 (s); LC-MS (ESI+)m/z 327.08 (M-morpholine); HRMS (ESI+) m/z calculated for C₂₁H₂₂ClFN₅O(M+H)⁺ 414.1491. found 414.1497 (M+H)⁺, 327.0819 (M—morpholine).

N⁴-(2-Chlorophenyl)-N²-[4-N-(2-hydroxyethyl)carbamoylphenyl]pyrimidine-2,4-diamine(9l, method t in FIG. 7A)

A mixture of chloropyrimidine 2l (0.100 g, 0.416 mmol) and4-amino-N-(2-hydroxyethyl)benzamide (0.075 g, 0.416 mmol) in HCl (1 mLof 0.1N aq.) was heated in a microwave reactor at 140° C. for 30 min.The contents were then stirred in the microwave vial at room temperaturefor 30 min. The mixture was filtered and the obtained solid was washedwith water (20 mL) and sodium bicarbonate (sat. aq. 50 mL). The crudeproduct was purified by chromatography (silica gel, hexane: ethylacetate) to provide 9l as an off-white solid (0.087 g, 54%). m.p.171-173° C., HPLC 100% [R_(t)=8.8 min, 40% MeOH, 60% water (with 0.1%TFA) 20 min.]; ¹H NMR (400 MHz, MeOH-d₄), δ 8.28 (t, J=6.2 Hz, 1H, peakdisappeared upon D₂O shake), 8.00 (d, J=5.9 Hz, 1H), 7.84 (dd, J=8.0,1.5 Hz, 1H), 7.71-7.64 (m, 4H), 7.50 (dd, J=8.0, 1.5 Hz, 1H), 7.37 (td,J=8.0, 1.5 Hz, 1H), 7.22 (td, J=8.0, 1.5 Hz, 1H), 6.30 (d, J=5.9 Hz,1H), 3.70 (t, J=5.5 Hz, 2H), 3.49 (q, J=5.5 Hz, 2H); ¹³C NMR (100 MHz,DMSO-d₆), δ 172.68, 166.63, 161.90, 159.62, 156.84, 144.10, 136.56,130.31, 128.61, 128.27, 128.07, 126.92, 126.82, 117.93, 99.18, 60.56,42.68; LC-MS (ESI+) m/z 384.12 (M+H)⁺; HRMS (ESI+) m/z calculated forC₁₉H₁₉ClN₅O₂ (M+H)⁺ 384.1222. found 384.1219.

N⁴-(2-Chlorophenyl)-N²-(4-carbamoyl)-5-fluoropyrimidine-2,4-diamine (9m,method v in FIG. 7A)

A mixture of chloropyrimidine 2k (0.500 g, 1.935 mmol), 4-aminobenzamide(0.265 g, 1.935 mmol) in methanol (3 mL), was heated in 5 ml sealedpressure tube at 100° C. for 6 hrs. The white precipitate was isolatedby filtration and washed with methanol (2 mL). The white solid wassonicated for 5 min. in sodium bicarbonate (aq. sat., 10 mL). Themixture was filtered and the solid washed with water (10 mL) and finallywith methanol (2 mL) to provide 9m (0.439 g, 63%) as an off-whitepowder. m.p. 250-252° C. HPLC 100% [R_(t)=7.8 min, 50% MeOH, 50% water(with 0.1% formic acid) 20 min.]; ¹H NMR (400 MHz, DMSO-d₆), δ 9.42 (s,1H), 9.31 (s, 2H), 8.12 (d, J_(H-F)=3.6 Hz, 1H), 7.70 (s, 1H), 7.59 (dd,J=8.6, 1.3 Hz, 1H), 7.56 (dd, J=8.6, 1.3 Hz) overlapping 7.54 (d, J=8.7Hz, 2H), 7.48 (d, J=8.7 Hz, 2H), 7.42 (td, J=7.7, 1.5 Hz, 1H), 7.35 (td,J=7.6, 1.6 Hz, 1H), 7.08 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.57,155.66 (d, J_(C-F)=3.0 Hz), 151.35 (d, J_(C-F)=11.7 Hz), 144.27, 142.54,141.66 (d, J_(C-F)=19.0 Hz), 140.09, 135.77, 131.27, 130.41, 129.79,128.67, 128.41, 126.18, 117.31; ¹⁹F NMR (376 MHz, DMSO-d₆), δ −165.45(bd, J_(H-F)=3.4 Hz) LC-MS (ESI+) m/z 358.09 (M+H)⁺; HRMS (ESI+) m/zcalculated for C₁₇H₁₄ClFN₅O (M+H)⁺ 358.0865. found 358.0868.

N⁴-(2-Chlorophenyl)-MN-(4-aminosulfonyl)-5-fluoropyrimidine-2,4-diamine(9n, method u in FIG. 7A)

A mixture of chloropyrimidine 2k (free base) (0.100 g, 0.387 mmol),4-aminobenzenesulfonamide (0.066 g, 0.0.387 mmol), X-Phos(2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl) (0.019 g, 0.0387mmol), bis(dibenzylideneacetone) palladium(0) (0.022 g, 0.0387 mmol),potassium carbonate (0117 g, 0.851 mmol) in tert-BuOH (2.0 mL) washeated in 5 ml sealed pressure tube at 100° C. for 64 hours under argon.The solid was isolated by filtration and suspended in ethyl acetate (5mL) and sonicated for 5 minutes. The mixture was filtered and the solidwashed with ethyl acetate (5 mL). The crude product was dissolved inDMSO (2 mL) and filtered to remove undissolved material. The DMSO wasremoved in vacuo at 50° C. to provide the title sulfonamide 9n (0.080 g,53%) as a brown solid. m.p. 253-255° C. HPLC 99% [R_(t)=11.2 min, 40%MeOH, 60% water (with 0.1% TFA) 66 min.]; ¹H NMR (400 MHz, DMSO-d₆), δ9.57 (s, 1H), 9.39 (s, 1H), 8.13 (d, J_(H-F)=3.7 Hz, 1H), 7.61-7.52 (m,4H), 7.46-7.38 (m, 3H), 7.35 (t, J=8.0 Hz, 1H), 7.07 (s, 1H). ¹H NMR(400 MHz, DMSO-d₆+D₂O), δ 8.09 (d, J_(H-F)=3.5 Hz, 1H), 7.58 (dd, J=7.9,1.5 Hz, 1H), 7.54-7.48 (m, 3H), 7.44-7.39 (m, 3H), 7.35 (dt, J=7.4, 1.7Hz, 1H); ¹⁹F NMR (376 MHz, DMSOd₆-+D₂O), δ −164.56 (d, J=3.1 Hz); ¹³CNMR (100 MHz, DMSO-d₆) 155.67 (d, J_(C-F)=2.8 Hz), 151.57, 151.46,144.66, 142.73, 141.79, 141.60, 140.27, 136.06, 135.87, 131.65, 130.42,130.19, 128.42, 126.85, 117.46. LC-MS (ESI+) m/z 394.05 (M+H)⁺; HRMS(ESI+) m/z calculated for C₁₆H₁₄ClFN₅O₂S (M+H)⁺ 394.0535. found394.0537.

N⁴-(2-Chlorophenyl)-N²-[4-(2H-tetrazol-5-yl)phenyl]pyrimidine-2,4-diaminehydrochloride (12a, method a in FIG. 8)

A mixture of 2l (0.072 g, 0.299 mmol) and4-(2H-tetrazol-5-yl)phenylamine (11a) (0.050 g, 0.310 mmol) in EtOH (2.0mL) was heated in a microwave reactor at 150° C. for 40 min. Theprecipitate obtained upon cooling the reaction mixture was filtered andwashed with EtOH (5 mL) to provide 12a (0.095 g, 79%) as a yellow solid(¹H NMR analysis indicated the presence of trace impurities). Theproduct was further purified by washing with methanol (5 mL) to givepure 12a (0.045 g, 37%) as a yellow solid. m.p. 250° C. (dec.). HPLC100% (R_(t)=6.21 min., 50% MeOH in 0.1% TFA water, 20 min.); ¹H NMR (400MHz, DMSO-d₆) δ 10.76 (s, 1H), 10.64 (s, 1H), 8.09 (d, J=6.9 Hz, 1H),7.86 (d, J=8.4 Hz, 2H), 7.69-7.59 (m, 4H), 7.48 (t, J=7.4 Hz, 1H), 7.41(t, J=7.5 Hz, 1H), 6.52 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.07,153.08, 146.47, 140.76, 134.74, 130.64, 130.17, 129.48, 129.32, 129.31,128.57, 128.21, 121.13, 119.58; elemental analysis calculated forC₁₇H₁₄Cl₂N₈: C, 50.89; H, 3.52; N, 27.93. Found: C, 51.22; H, 3.50; N,27.54; LC-MS (ESI−) m/z 363.08 (M−H−HCl)⁻; HRMS (ESI−) m/z calculatedfor C₁₇H₁₂ClN₈ (M−H−HCl)⁻ 363.0879. found 363.0871.

N⁴-(2-Chlorophenyl)-N²-[4-(2H-tetrazol-5-yl)phenyl]-5-fluoropyrimidine-2,4-diaminehydrochloride (12b, method a in FIG. 8)

A mixture of 2k (free base) (0.076 g, 0.298 mmol) and4-(2H-tetrazol-5-yl)phenylamine (11a) (0.050 g, 0.310 mmol) in EtOH (2.0mL) was heated in a microwave reactor at 170° C. for 40 min. Theprecipitate obtained upon cooling the reaction mixture was filtered andwashed with EtOH (5.0 mL) to get 12b (0.090 g, 72%) as a brown-yellowsolid. The ¹H NMR spectrum showed the presence of a baseline impurityand the product was further purified by washing with methanol (5 mL) toprovide pure 12b (0.041 g, 33%) as a brown-yellow solid. m.p.: 223° C.(dec.). HPLC 99% (R_(t)=10.37 min., 50% MeOH in 0.1% TFA water, 20min.); ¹H NMR (400 MHz, DMSO-d₆) δ 9.98 (s, 1H), 9.91 (s, 1H), 8.25 (d,J=4.0 Hz, 1H), 7.74 (d, J=8.7 Hz, 2H), 7.64 (d, J=7.8 Hz, 1H), 7.62-7.56(m, 3H), 7.50-7.38 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 155.74 (d,J=3.0 Hz), 151.50 (d, J=11.7 Hz), 144.37, 141.45 (d, J=247.45 Hz),141.68 (d, J=17.6 Hz), 136.06, 131.58, 130.43, 130.15, 128.41, 127.85,118.47. ¹⁹F NMR (376 MHz, DMSO) δ −165.12 (s); elemental analysiscalculated for C₁₇H₁₃Cl₂FN₈: C, 48.70; H, 3.13; N, 26.73. Found: C,49.09; H, 3.13; N, 26.48. LC-MS (ESI−) m/z 381.1 (M−H)⁻; HRMS (ESI−) m/zcalculated for C₁₇H₁₁ClFN₈ (M−H)⁻ 381.0785. found 381.0784.

N⁴-(2-Chlorophenyl)-N²-[3-hydroxy-4-(2H-tetrazol-5-yl)phenyl]-5-fluoropyrimidine-2,4-diaminehydrochloride (12c, method a FIG. 8)

A mixture of 2k (freebase) (0.072 g, 0.279 mmol) and tetrazole 11b(0.050 g, 0.282 mmol) in EtOH:HCl (1:1, 2.0 mL, 0.1 M HCl) was heated ina microwave reactor at 160° C. for 40 min. The precipitate obtained uponcooling was filtered and rinsed with EtOH (5 mL) to obtain the productwith base-line impurity (0.096 g, 77%) as a yellow solid. This solid wasfurther purified by washing with methanol (5 mL) to provide pure 12c(0.044 g, 36%) as a yellow solid. m.p. 214° C. (dec.). HPLC 93%(R_(t)=12.99 min., 50% MeOH in 0.1% TFA water, 20 min.); ¹H NMR (400MHz, DMSO-d₆) δ 10.66 (s, 1H), 9.44 (s, 1H), 9.24 (s, 1H), 8.13 (d,J=3.3 Hz, 1H), 7.66-7.54 (m, 4H), 7.41 (t, J=7.5 Hz, 1H), 7.33 (t, J=7.4Hz, 1H), 7.29-7.23 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) 156.34, 155.80(d, J=3.1 Hz), 151.46 (d, J=11.5 Hz), 145.58, 141.61 (d, J=247.2 Hz),141.23 (d, J=19.1 Hz), 135.90, 131.04, 130.42, 129.71, 129.25, 128.38,128.20, 110.49, 105.32, 103.39; ¹⁹F NMR (376 MHz, DMSO-d₆) δ −165.94(s); LC-MS (ESI−) m/z 397.07 (M−H)⁻; HRMS (ESI−) m/z calculated forC₁₇H₁₁ClFN₈O (M−H)⁻ 397.0734. found 397.0730.

N⁴-(2-Chlorophenyl)-N²-[4-(2H-tetrazol-5-yl)phenyl]-5-chloropyrimidine-2,4-diaminehydrochloride (12d, method a FIG. 8)

A mixture of chloropyrimidine 2o (0.093 g, 0.3 mmol) and4-(2H-tetrazol-5-yl)phenylamine (11a) (50 mg, 0.3 mmol) in EtOH (2 mL)was heated in a microwave reactor at 150° C. for 40 min. The precipitateobtained upon cooling was filtered and washed with EtOH (5 mL) toprovide impure 12d (105 mg, 81%) as a yellow color solid. Analysis ofthe ¹H NMR spectrum indicated the presence of trace impurities. Theproduct was further purified by washing with hot ethyl acetate (5 mL)and hot methanol (5 mL) to provide pure 12d (0.032 g, 25%) as a yellowsolid. m.p. 238° C. (dec.). HPLC 91% (R_(t)=5.71 min., 70% MeOH in 0.1%TFA water, 20 min.); ¹H NMR (400 MHz, DMSO-d₆) δ 9.72 (s, 1H), 9.05 (s,1H), 8.19 (s, 1H), 7.71-7.56 (m, 6H), 7.52-7.38 (m, 2H); ¹³C NMR (100MHz, DMSO-d₆) δ 157.73, 157.53, 154.65, 143.69, 136.39, 131.71, 130.38,130.32, 128.69, 128.49, 127.87, 119.04, 104.93; LC-MS (ESI−) m/z 397.05(M−H)⁻; HRMS (ESI−) m/z calculated for C₁₇H₁₁Cl₂N₈ (M−H)⁻ 397.0489.found 397.0472.

1-18. (canceled)
 19. A method of treating cancer, comprising the step ofadministering to a patient in need thereof a compound of the followingformula:

wherein R¹ is selected from the group consisting of H, Cl, F, Br, I,C₁-C₆ alkyl, CN, NO₂, and NH₂; R² is H, F, or Cl; each R³ is 2-Cl; andeach R⁴ is selected, independently, from the group consisting of H,COOH, CONH₂, CONHR⁵, SO₂NH₂, CONHSO₂R⁵, tetrazole, 4-morpholine, orCOR⁵, wherein R⁵ is C₁-C₆ alkyl, cycloalkyl, heteroaryl, or heteroalkyl,n is 1, and m is 1-5, or a pharmaceutically acceptable salt thereof. 20.The method of claim 19, wherein the cancer is characterized by Aurorakinase up-regulation.
 21. The method of claim 19, wherein the compoundis a selective inhibitor of Aurora A kinase over Aurora B kinase. 22.The method of claim 19, wherein the compound is a DFG-out inhibitor. 23.The method of claim 19, wherein the cancer is selected from the groupconsisting of breast cancer and colorectal cancer.
 24. The method ofclaim 19, wherein the compound has the following formula:

wherein R¹ is selected from the group consisting of H, Cl, F, Br, I, CH₃and NH₂; R² is H, F, or Cl; and R⁴ is selected from the group consistingof H, COOH, 2-CONH₂, 4-CONH₂, SO₂NH₂, tetrazole, and 4-morpholine. 25.The method of claim 19, wherein R⁴ is 4-COOH, and m is
 1. 26. The methodof claim 19, wherein m is 1, and R⁴ is COOH, COR⁵, CONH₂, CONHR⁵, orCONHSO₂R⁵, wherein R⁵ is C₁-C₆ alkyl, cycloalkyl, heteroaryl, orheteroalkyl.
 27. A method of treating cancer, comprising the step ofadministering to a patient in need thereof a compound of the followingformula:

wherein R¹ is H, R³ is ortho-Cl, R⁴ is para-CH₂—COOH, and R⁵ is H; R¹ isH, R³ is ortho-Cl, R⁴ is para-COOH and meta-OH, and R⁵ is H; R¹ is H, R³is ortho-Cl, R⁴ is meta-COOH, and R⁵ is H; R¹ is H, R³ is ortho-Cl, R⁴is para-CONH₂, and R⁵ is H; R¹ is H, R³ is ortho-Cl, R⁴ is para-COOH,and R⁵ is CH₃; R¹ is H, R³ is ortho-Cl, R⁴ is para-COOH, and R⁵ isCH₃—CH₂; R¹ is F, R³ is ortho-Cl, R⁴ is meta-COOH, and R is H; R¹ is H,R³ is ortho-Cl, R⁴ is para-COOH and meta OH; R¹ is H, R³ is ortho-Cl, R⁴is para-COOH, and R⁵ is H; R¹ is F, R³ is ortho-Cl, R⁴ is para-COOH, andR⁵ is H; R¹ is F, R³ is ortho-Cl, R⁴ is H, and R is H; or R¹ is H, R³ isortho-Cl, R⁴ is para-OCH₃, and R⁵ is H.
 28. The method of claim 27,wherein the cancer is selected from the group consisting of breastcancer and colorectal cancer.
 29. A method of treating cancer,comprising the step of administering to a patient in need thereof acompound chosen from the follow group:


30. The method of claim 29, wherein the cancer is selected from thegroup consisting of breast cancer and colorectal cancer.